LIBRARY 

OF  THE 

UNIVERSITY  OF  CALIFORNIA. 


OF    CALIFOKNI* 


Class 


ELEMENTS 


OF 


WATER  BACTERIOLOGY 


WITH  SPECIAL  REFERENCE  TO 


SANITARY   WATER  ANALYSIS. 


BY 

SAMUEL  GATE   PRESCOTT, 

Assistant  Professor  of  Industrial  Biology, 
AND 

CHARLES-EDWARD  AMORY  WINSLOW, 

Assistant  Professor  of  Sanitary  Biology, 

IN  THE 
MASSACHUSETTS  INSTITUTE  OF  TECHNOLOGY.         , 


UNIVERSITY 

OF 
SdUFOR 


SECOND   EDITION,   REWRITTEN 
FIRST    THOUSAND. 


NEW  YORK: 

JOHN  WILEY   &   SONS. 

LONDON:   CHAPMAN   &   HALL,  LIMITED. 

1908. 


PS 


Library 


v^- 


Copyright,  1904,  1908, 

BY 

S.  C.  PRESCOTT 

ANU 

C.-E.  A.  WINSLOW 


Stanhope  ipress 

F.    H.   GILSON     COMPANY 
BOSTON.     U.S.A. 


DEDICATED 
TO 


BY  TWO  OF  HIS   PUPILS, 
AS  A    TOKEN    OF    GRATEFUL    AFFECTION. 


201695 


PREFACE  TO  FIRST  EDITION 


THE  general  awakening  of  the  community  to  the 
importance  of  the  arts  of  sanitation  —  accelerated  by  the 
rapid  growth  of  cities  and  the  new  problems  of  urban 
life  —  demands  new  and  accurate  methods  for  the  study 
of  the  microbic  world.  Bacteriology  has  long  since 
ceased  to  be  a  subject  of  interest  and  importance  to  the 
medical  profession  merely,  but  has  become  intimately 
connected  with  the  work  of  the  chemist,  the  biologist, 
and  the  engineer.  To  the  sanitary  engineer  and  the 
public  hygienist  a  knowledge  of  bacteriology  is  indis- 
pensable. 

In  the  swift  development  of  this  science  during  the 
last  ten  years  perhaps  no  branch  of  bacteriology  has 
made  more  notable  progress  than  that  which  relates  to 
the  sanitary  examination  of  water.  After  a  brief  period 
of  extravagant  anticipation,  and  an  equally  unreason- 
able era  of  neglect  and  suspicion,  the  methods  of  the 
practical  water  bacteriolpgist  have  gradually  made  their 
way,  until  it  is  recognized  that,  on  account  of  their  deli- 
cacy, their  directness,  and  their  certainty,  these  methods 
now  furnish  the  final  criterion  of  the  sanitary  condition 
of  a  potable  water. 


vi  Preface 

A  knowledge  of  the  new  science  early  became  so 
indispensable  for  the  sanitary  expert  that  a  special 
course  in  the  Bacteriology  of  Water  and  Sewage  has  for 
some  years  been  given  to  students  of  biology  and  sani- 
tary engineering  in  the  Biological  Department  of  the 
Massachusetts  Institute  of  Technology.  For  workers  in 
this  course  the  present  volume  has  been  especially 
prepared,  and  it  is  fitting,  we  think,  that  such  a  manual 
should  proceed  from  an  institution  whose  faculty,  gradu- 
ates, and  students  have  had  a  large  share  in  shaping  the 
science  and  art  of  which  it  treats.  We  shall  be  grati- 
fied, however,  if  its  field  of  usefulness  extends  to  those 
following  similar  courses  in  other  institutions,  or  occupied 
professionally  in  sanitary  work. 

The  treatment  of  the  subject  in  the  many  treatises  on 
General  Bacteriology  and  Medical  Bacteriology  is  neither 
special  enough  nor  full  enough  for  modern  needs.  The 
classic  work  of  Grace  and  Percy  Frankland  is  now  ten 
years  old;  and  even  Horrocks'  valuable  " Bacteriological 
Examination  of  Water  "  requires  to  be  supplemented  by 
an  account  of  the  developments  in  quantitative  analysis 
which  have  taken  place  on  this  side  of  the  Atlantic. 

It  is  for  us  a  matter  of  pride  that  Water  Bacteriology 
owes  much  of  its  value,  both  in  exactness  of  method 
and  in  common-sense  interpretation,  to  American  sani- 
tarians. The  English  have  contributed  researches  of  the 
greatest  importance  on  the  significance  of  certain  intes- 
tinal bacteria;  but  with  this  exception  the  best  work 


Preface  vii 

on  the  bacteriology  of  water  has,  in  our  opinion,  been 
done  in  this  country.  Smith,  Sedgwick,  Fuller,  Whipple, 
Jordan,  and  their  pupils  and  associates  (not  to  mention 
others)  have  been  pioneers  in  the  development  of  this 
new  field  in  sanitary  science.  To  gather  the  results  of 
their  work  together  in  such  form  as  to  give  a  correct 
idea  of  the  best  American  practice  is  the  purpose  of 
this  little  book;  and  this  we  have  tried  to  do,  with  such 
completeness  as  shall  render  the  volume  of  value  to  the 
expert  and  at  the  same  time  with  such  freedom  from 
undue  technicality  as  to  make  it  readable  for  the  layman. 
It  should  be  distinctly  understood  that  students  using 
it  are  supposed  to  have  had  beforehand  a  thorough 
course  in  general  bacteriology,  and  to  be  equipped  for 
advanced  work  in  special  lines. 

BOSTON,  March  10, 


PREFACE  TO  SECOND  EDITION 


SINCE  the  appearance  of  the  first  edition  of  this  work 
the  investigation  of  various  phases  of  the  bacteriology  of 
water  has  been  advanced  with  zeal  by  many  workers  on 
both  sides  of  the  Atlantic.  As  a  result  a  considerable 
mass  of  new  evidence  has  accumulated  which  has  estab- 
lished the  bacteriological  examination  of  water  on  a 
firmer  basis  than  ever,  and  shown  it  to  be  the  most  direct, 
accurate  and  practical  method  at  the  disposal  of  the 
sanitarian. 

The  study  of  presumptive  tests  for  the  colon  bacillus 
has  advanced,  with  highly  satisfactory  results.  The 
methods  for  the  isolation  of  specific  pathogenic  organ- 
isms have  also  been  notably  improved. 

An  excellent  volume  on  Water  Bacteriology  by  Dr. 
W.  G.  Savage  has  appeared  during  the  past  year,  which 
shows  the  English  methods  of  investigation  and  interpre- 
tation to  be  closely  in  accord  with  those  used  in  America. 

It  has  been  our  aim  in  preparing  this  new  edition  to 
include  the  results  of  the  work  of  the  last  four  years 
which  bear  on  the  practical  investigation  of  sanitary 
questions  connected  with  water  supply.  Considerable 
additions  have  been  made  to  the  treatment  of  the  prob- 

ix 


x  Preface 

lems  of  self  purification  in  Chapter  I,  to  the  description 
of  methods  for  the  isolation  of  the  typhoid  bacillus  in 
Chapter  V,  to  the  treatment  of  the  interpretation  of  the 
colon  test  in  Chapter  VII,  to  an  account  of  the  newer 
presumptive  tests  for  B.  coli  in  Chapter  VIII,  and  to 
the  discussion  of  the  significance  of  intestinal  bacteria, 
other  than  B.  coli,  in  Chapter  IX.  A  new  chapter  has 
been  introduced  on  The  Bacteriology  of  Sewage  and 
Sewage  Effluents,  in  recognition  of  the  growing  impor- 
tance of  this  branch  of  the  subject. 

THE  BIOLOGICAL  LABORATORIES, 

MASSACHUSETTS  INSTITUTE  OF  TECHNOLOGY, 

Boston,  January  i,  igo8. 


TABLE  OF  CONTENTS. 


CHAPTER  I. 

THE  BACTERIA  IN  NATURAL  WATERS i 


CHAPTER  II. 
THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION  OF  WATER  .       25 

CHAPTER  III. 

THE  INTERPRETATION  OF  THE  QUANTITATIVE  BACTERIOLOGICAL 
EXAMINATION 46 

CHAPTER   IV. 

DETERMINATION  OF  THE  NUMBER  OF   ORGANISMS  DEVELOPING 
AT  THE  BODY  TEMPERATURE 57 

CHAPTER    V. 
THE  ISOLATION  OF  SPECIFIC  PATHOGENES  FROM  WATER     ...       67 

CHAPTER  VI. 
METHODS  FOR  THE  ISOLATION  OF  THE  COLON  BACILLUS     ...      86 

CHAPTER   VII. 
SIGNIFICANCE  OF  THE  PRESENCE  OF  B.  COLI  IN  WATER     ...     112 

CHAPTER   VIII. 

PRESUMPTIVE  TESTS  FOR  B.  COLI 141 

xi 


xii  Table  of  Contents. 

CHAPTER   IX. 

PAGE. 

OTHER  INTESTINAL  BACTERIA 152 

CHAPTER   X. 

THE  SIGNIFICANCE  AND  APPLICABILITY  OF  THE  BACTERIOLOGICAL 
EXAMINATION 170 

CHAPTER  XI. 
THE  BACTERIOLOGY  OF  SEWAGE  AND  SEWAGE  EFFLUENTS      .     184 

APPENDIX 203 

REFERENCES 217 

INDEX 247 


ELEMENTS  OF  WATER  BACTERIOLOGY 


CHAPTER   I. 

THE   BACTERIA  IN  NATURAL  WATERS. 

BACTERIA  are  the  most  numerous  and  the  most  widely 
distributed  of  living  things.  They  are  present  not  merely 
at  the  surface  of  the  earth  or  in  the  bodies  of  water  which 
partially  cover  it,  as  is  the  case  with  most  other  living 
things,  but  in  the  soil  itself,  and  in  the  air  above,  and  in 
the  waters  under  the  earth. 

Probably  no  organisms  are  more  sensitive  to  external 
conditions,  and  none  respond  more  quickly  to  slight 
changes  in  their  environment.  Temperature,  moisture, 
and  oxygen  are  of  importance  in  controlling  their  distri- 
bution; but  the  most  significant  factor  is  the  amount  of 
food  supply.  Bacteria  and  decomposing  organic  matter 
are  always  associated,  and  for  this  reason  a  brief  consid- 
eration of  the  general  relation  of  bacteria  to  their  sources 
of  food  supply  must  precede  the  study  of  their  distribution 
in  any  special  medium. 

The  bacteria  possess  greater  constructive  ability  than 
any  animal  organisms.  They  lack,  however,  the  power  of 


2  Elements  of  Water  Bacteriology. 

green  plants  to  build  up  their  own  food  from  compounds 
like  carbon  dioxide  and  nitrates  which  have  no  stored 
potential  energy.  The  food  requirements  of  various 
bacterial  types  differ,  however,  widely  among  themselves. 
Fischer  (1900)  has  divided  the  whole  group  into  three 
great  subdivisions  according  to  the  nature  of  their  metab- 
olism. The  Prototrophic  forms  are  characterized  by 
minimal  nutrient  requirements,  including  organisms  like 
the  nitrifying  bacteria  which  require  no  organic  compounds 
at  all  but  derive  their  nourishment  from  carbon  dioxide 
or  carbonates,  nitrites  and  phosphates,  or  from  inorganic 
ammonium  salts.  A  second  group,  the  Metatrophic 
bacteria,  includes  those  forms  which  require  organic 
matter,  nitrogenous  and  carbonaceous,  but  are  not  depen- 
dent on  the  fluids  of  the  living  plant  or  animal.  Finally, 
the  Paratrophic  bacteria  are  the  true  parasites,  which 
exist  only  within  the  living  tissues  of  other  organisms. 
These  subdivisions,  like  all  groups  among  the  lower  plants, 
are  not  sharply  defined;  and  the  metatrophic  bacteria 
in  particular  exhibit  every  gradation,  from  types  which 
grow  in  water  with  a  trace  of  free  ammonia,  to  organisms 
like  the  colon  bacillus  which  normally  occur  on  the 
surface  of  the  plant  or  animal  body,  feeding  upon  the 
fluids  or  on  the  extraneous  material  collected  upon  its 
surface. 

The  vast  majority  of  bacteria  belong  to  the  second,  or 
metatrophic  group,  living  as  saprophytes  on  dead  organic 
matter  wherever  it  may  occur  in  nature,  and  particularly 


The  Bacteria  in  Natural   Waters.  3 

in  that  diffuse  layer  of  decomposing  plant  and  animal 
material  which  we  call  the  humus,  or  surface  layer  of 
the  soil.  Wherever  there  is  life,  waste  matter  is  constantly 
being  produced,  and  this  finds  its  way  to  the  earth  or  to 
some  body  of  water.  The  excretions  of  animals,  the 
dead  tissues  and  broken-down  cells  of  both  animals  and 
plants,  as  well  as  the  wastes  of  domestic  and  industrial 
life,  all  eventually  find  their  way  to  the  soil.  In  a  majority 
of  cases  these  substances  are  not  of  such  chemical  com- 
position that  they  can  be  utilized  at  once  by  green  plants 
as  food,  but  it  is  first  necessary  that  they  go  through  a 
fermentation  or  transformation  in  which  their  chemical 
composition  becomes  changed;  and  it  is  as  the  agents  of 
this  transformation  that  bacteria  assume  their  greatest 
importance  in  the  world  of  life. 

We  may  take  the  decomposition  of  a  comparatively 
simple  excretory  product,  urea,  as  an  example  of  the  part 
which  the  bacteria  play  in  the  preparation  of  plant  food. 
Through  the  activity  of  an  enzyme  produced  by  certain 
bacteria  this  compound  unites  with  two  molecules  of  water 
and  is  converted  into  ammonium  carbonate, 

NH2 

CO  +  2H20  =  (NH4)2C03. 

XNH2 

This,  however,  is  only  part  of  the  process.  While  green 
plants  can  derive  their  necessary  nitrogen  in  part,  at  least, 
from  ammonium  compounds,  it  is  a  well-established  fact 
that  this  element  may  be  obtained  much  more  readily 


4  Elements  of  Water  Bacteriology. 

from  nitrates,  and  there  are  other  bacteria  which  as  a 
further  step  oxidize  the  ammoniacal  nitrogen  to  a  more 
available  form.  This  process  of  oxidation  is  known  as 
nitrification,  and  takes  place  in  a  succession  of  steps,  the 
organic  nitrogen  being  first  converted  to  the  form  of 
ammonium  salts,  and  these  in  turn  to  nitrites  and  nitrates, 
the  oxygen  used  coming  from  the  air.  Several  groups  of 
organisms  are  instrumental  in  bringing  about  this  con- 
version. It  is  generally  assumed  that  one  group  attacks 
the  ammonium  compounds  and  changes  them  to  nitrites, 
while  another  group  completes  the  oxidation  to  nitrates. 
In  the  latter  form  nitrogen  is  readily  taken  up  by  green 
plants  to  be  built  up  into  more  complex  albuminoid  sub- 
stances (organic  nitrogen)  through  the  constructive  power 
of  chlorophyll. 

This  never-ending  cycle  is  illustrated  in  the  accom- 
panying figure,  devised  by  Sedgwick  (Sedgwick,  1889)  to 
illustrate  the  transformations  of  organic  nitrogen  in  nature, 
the  increasing  size  and  closeness  of  the  spiral  on  the  left- 
hand  side  indicating  the  progressive  complexity  of  organic 
matter  as  built  up  by  the  chlorophyll  bodies  of  green 
plants  in  the  sunlight,  and  the  other  half  of  the  figure  the 
reverse  process  carried  out  largely  by  the  bacteria.  In 
nature  there  are  many  short  circuits,  as,  for  instance,  when 
dead  organic  matter  is  used  as  food  for  animals  and  built 
up  into  the  living  state  again  without  being  nitrified  and 
acted  upon  by  green  plants;  but  the  complete  cycle  of 
organic  nitrogen  is  as  indicated  on  the  diagram. 


The  Bacteria  in  Natural   Waters. 


We  have  dwelt  thus  at  length  upon  the  general  relation 
between  bacteria  and  organic  decomposition  because  in 
this  relation  will  be  found  the  master  key  to  the  distri- 
bution of  bacteria  in  water  as  well  as  in  other  natural 
habitats.  It  is  true  that  certain  peculiar  forms  may  at 
times  multiply  in  fairly  pure  waters;  but  in  general  large 
numbers  of  bacteria  are  found  only  in  connection  with 
the  organic  matter  upon  which  they  feed.  Such  organic 


(        NITROGEN  A3 
<  ORGANIC  NITROGEN 
/(ALBUMINOID  AMMONIA) 


THE  SPHERE 

OF 
ORGANISMS 

AND 
THE  HISTORY 

OF 
ORGANIC  MATTEfi. 


FIG.  i. 

matter  is  particularly  abundant  in  the  surface  layer  of  the 
soil.  Here,  therefore,  the  bacteria  are  most  numerous; 
and  in  other  media  their  numbers  vary  according  to  the 
extent  of  contact  with  the  living  earth. 

Natural  waters  group  themselves  from  a  bacteriological 
standpoint  in  four  well-marked  classes,  according  to  their 


6  Elements  of  Water  Bacteriology. 

relation  to  the  rich  layers  of  bacterial  growth  upon  the 
surface  of  the  globe.  There  are  first  the  atmospheric- 
waters  which  have  never  been  subject  to  contact  with 
the  earth;  second,  the  surface-waters  immediately  exposed 
to  such  contamination  in  streams  and  pools;  third,  the 
lakes  and  large  ponds  in  which  storage  has  reduced 
bacterial  numbers  to  a  state  of  comparative  purity; 
and  fourth,  the  ground-waters  from  which  previous  con- 
tamination has  been  even  more  completely  removed  by 
filtration  through  the  deeper  layers  of  the  soil. 

Even  rain  and  snow,  the  original  sources  of  our  potable 
waters,  are  by  no  means  free  from  germs,  but  contain 
them  in  numbers  varying  according  to  the  amount  of  dust 
present  in  the  air  at  the  time  of  the  precipitation.  After 
a  long-continued  storm  the  atmosphere  is  washed  nearly 
free  of  bacteria,  so  that  a  considerable  series  of  sterile 
plates  may  often  be  obtained  by  plating  i-c.c.  samples. 
These  results  are  in  harmony  with  the  observations  of 
Tissandier  (reported  by  Duclaux,  1897),  who  found  that 
the  dust  in  the  air  amounted  to  23  mg.  per  cubic  meter 
in  Paris  and  4  mg.  in  the  open  country.  After  a  rain- 
fall these  figures  were  reduced  to  6  mg.  and  .25  mg., 
respectively. 

With  regard  to  what  may  be  considered  normal  values 
for  rain,  satisfactory  data  are  not  abundant.  Those 
obtained  by  Miquel  (Miquel,  1886),  during  the  period 
1883-1886,  showed  on  the  average  4.3  bacteria  per  c.c.  in 
the  country  (Montsouris)  and  19  per  c.c.  in  Paris.  Snow 


The  Bacteria  in  Natural  Waters  7 

shows  rather  higher  numbers  than  rain.  Janowski 
(Janowski,  1888)  found  in  freshly  fallen  snow  from  34  to 
463  bacteria  per  c.c.  of  snow-water. 

As  soon  as  the  raindrop  touches  the  surface  of  the 
earth  its  real  bacterial  contamination  begins.  Rivulets 
from  ploughed  land  or  roadways  may  often  contain 
several  hundred  thousand  bacteria  to  the  cubic  centi- 
meter; and  furthermore  the  amounts  of  organic  and 
mineral  matters  which  serve  as  food  materials,  and  thus 
become  a  factor  in  later  multiplication  of  organisms,  are 
greatly  increased. 

In  the  larger  streams  several  conditions  combine  to 
make  these  enormous  bacterial  numbers  somewhat  lower. 
Ground-water  containing  little  microbic  life  enters  as  a 
diluting  factor  from  below.  The  larger  particles  of  organic 
matter  are  removed  by  sedimentation;  many  earth 
bacteria,  for  which  water  is  an  unfavorable  medium, 
gradually  perish;  and  in  general  a  new  condition  of 
equilibrium  tends  to  be  established.  It  is  difficult,  how- 
ever, to  find  a  river  in  inhabited  regions  which  does  not 
contain  several  hundreds  or  thousands  of  bacteria  to  the 
cubic  centimeter.  Furthermore,  heavy  rains  which  intro- 
duce wash  from  the  surrounding  watershed  may  at  any 
time  upset  whatever  equilibrium  exists,  and  surface- 
waters  are  apt  to  show  sudden  fluctuations  in  their  bac- 
terial content.  Particularly  in  the  spring  and  fall  high 
numbers  manifest  themselves,  and  seasonal  variations 
arise,  such  as  are  shown  in  the  appended  table. 


8 


Elements  of  Water  Bacteriology. 


SEASONAL  VARIATIONS  IN  BACTERIAL  CONTENT  OF 
RIVER  WATERS.  BACTERIA  PER  C.C.  MONTHLY 
AVERAGES. 


River. 

Year. 

Jan. 

Feb. 

Mar. 

Apr. 

May. 

June. 

Thames1      . 

1905-6 

2075 

1679 

1161 

277 

1064 

382 

Lea1     .   .   . 

" 

5J92 

3o83 

1308 

471 

135° 

598 

New1    .    .    . 

1455 

I3°4 

291 

149 

352 

198 

Mississippi2 

1900-01 

972 

2871 

i?95 

3597 

2152 

2007 

Potomac3     .     ' 

1906-7 

4400 

IOOO 

11,500 

3700 

75° 

2300 

Merrimac4  . 

1905 

14,200 

14,800 

10,300 

3600 

1900 

9600 

Susquehanna5 

1906 

95i° 

21,228 

3^326 

39>9°5 

6187 

2903 

River. 

Year. 

July. 

Aug. 

Sept. 

Oct. 

Nov. 

Dec. 

Thames1 
Lea1     .    . 

1905-6 

952 
1190 

... 

... 

... 

1633 
3946 

740 
2050 

New1    .    . 

450 

.  .  . 

.  .  . 

718 

621 

Mississippi2 

1900-01 

1832 

805 

.  .  . 

.  .  . 

202  1 

Potomac3 

1906-7 

2700 

3000 

6200 

2300 

1800 

6900 

Merrimac4 

I9°5 

3900 

19,500 

i3>5°° 

39,800 

8700 

.  .  . 

Susquehanna5 

1906 

685 

1637 

836 

7575 

26,224 

37>525 

Houston,  I9o6a,  I9o6b. 

New  Orleans,  1903. 

Figures  obtained  through  courtesy  of  F.  F.  Longley. 

Massachusetts,  1906. 

Harrisburg,  1907. 

In  general,  bacterial  numbers  are  highest  for  river- 
waters  in  the  winter  and  spring  months.  The  rainfall  is 
the  main  factor  which  affects  seasonal  variations,  but  its 
specific  effect  differs  with  different  streams.  The  imme- 
diate result  of  a  smart  shower  is  always  to  increase  con- 
tamination by  introducing  fresh  wash  from  the  surface 
of  the  ground.  More  prolonged  moderate  rain,  however, 


The  Bacteria  in  Natural  Waters. 


exerts  an  opposite  effect,  and  after  the  main  impurities 
which  can  be  washed  away  have  been  removed,  may 
dilute  the  stream  with  water  purer  than  itself.  In  the 
Kennebec  River  at  Waterville,  for  example,  Whipple 
(1907)  found  that  bacterial  counts  were  highest  at  the 
times  of  largest  stream  flow.  What  the  net  effect  of  rain 
may  be  depends  on  the  character  of  the  stream.  A  river 
of  fairly  good  quality  shows  its  highest  numbers  in  rainy 
periods.  With  a  highly  polluted  stream,  on  the  other 
hand,  the  constant  influx  of  sewage  overbalances  occa- 
sional contributions  of  surface  contamination.  Thus  Gage 
(1906)  shows  in  the  following  table  that  the  bacterial  con- 
tent of  the  Merrimac  is  highest  when  the  stream  is  lowest, 
that  is  when  its  sewage  content  is  least  subject  to  dilution. 

RELATION  BETWEEN  VOLUME  OF  FLOW  AND   BAC- 
TERIAL  CONTENT    IN    THE   MERRIMAC   RIVER. 
(GAGE,  1906.) 


Flow  of  Stream.     Cubic  feet  per 

Bacteria 

per  c.c. 

B.  coli. 

per  c.c. 

Watershed. 

Canal. 

Intake. 

Canal. 

Intake. 

Less  than  i 

7  COO 

10  800 

66 

88 

J—  2  .... 

6800 

6200 

CO 

CT 

2—4 

3600 

c6oo 

2O 

7Q 

34OO 

3100 

16 

2Q 

The  contrast  between  the  two  classes  of  riyers  is  well 
brought  out  in  a  study  of  the  Lahn  and  the  Wieseck,  by 
Kisskalt  (1906);  and  the  table  below,  compiled  from  his 
data,  gives  an  excellent  idea  of  the  total  numbers  of  bac- 


IO 


Elements  of  Water  Bacteriology. 


teria  and  their  seasonal  fluctuations  in  a  stream  of  fair 
quality  (the  Lahn)  and  a  highly  polluted  one  (the  Wieseck). 
In  the  former  case  the  bacterial  numbers  are  highest  when 
rain  brings  surface  pollution;  in  the  latter,  when  the  sewage 
constantly  present  is  least  diluted. 

MONTHLY    VARIATIONS    OF    BACTERIA    IN    A    NORMAL 
AND    A   POLLUTED    STREAM. 

(KISSKALT,   1906.) 


Date. 

Bacteria 

per  c.c. 

Date. 

Bacteria  p 

er  c.c. 

Lahn. 

Wieseck. 

Lahn. 

Wieseck. 

1904 

1904-5     ' 

July 

3i8 

104,000 

Dec.1 

1220 

21,200 

July 

132 

156,800 

Jan.1 

3668 

29,920 

Aug. 

840 

98,400 

Feb.1 

538o 

11,900 

Oct.1 

I235 

28,400 

Mar.1 

1210 

8250 

Oct.1 

420 

58,000 

Apr.1 

4925 

59io 

Nov. 

2340 

39,200 

May 

57° 

14,800 

Nov.1 

1740 

52,000 

June 

686 

50,180 

Dec.1 

780 

28,600 

1  Rain  or  high  water  due  to  previous  thaw. 

In  standing  waters  all  the  tendencies  which  make  for 
the  reduction  of  bacteria  are  intensified,  and  when  a  river 
passes  into  a  natural  or  artificial  reservoir  a  more  notable 
reduction  in  numbers  occurs.  The  following  table  shows 
the  striking  effect  produced  upon  the  water  of  the  Potomac 
River  by  its  successive  passage  through  the  three  reservoirs 
of  the  Washington  water  supply  in  the  first  nine  months 
of  1907.  We  owe  these  figures  to  the  courtesy  of  Mr.  F. 
F.  Longley,  the  engineer  in  charge  of  the  Washington 
filter  plant. 


The  Bacteria  in  Natural   Waters. 


II 


REDUCTION  OF  BACTERIA  IN  WASHINGTON  RESERVOIRS. 
BACTERIA  PER  C.C.  MONTHLY  AVERAGE,  1907. 


Potomac 

Dalecarlia 

Georgetown 

Washington 

Reservoir. 

Reservoir. 

Reservoir. 

City  Reservoir. 

Jan.            .    . 

4400 

2400 

2  20O 

95° 

Feb.            .    . 

1000 

95° 

1000 

75° 

Mar.           .    . 

11,500 

8300 

720O 

3600 

Apr 

3700 

2IOO 

I4OO 

47"> 

May 

o  /  WVJ 
75° 

35° 

325 

T"/  D 

130 

June 

2300 

95° 

600 

IOO 

July 

2700 

600 

35° 

1  60 

Aug. 

3000 

275 

425 

80 

Sept. 

6200 

1900 

230 

When  the  water  which  enters  a  pond  or  a  reservoir  has 
already  undergone  considerable  storage  and  reached  a 
comparatively  stable  condition,  the  diminution  due  to 
additional  storage  may  be  almost  negligible.  Thus 
Philbrick  (1905)  found  that  the  influent  water  of  the 
Chestnut  Hill  Reservoir  of  the  Metropolitan  Water  Works 
contained  on  the  average,  during  the  eleven  years,  1893- 
1903,  220  bacteria  per  c.c.,  and  the  effluent  179.  In  many 
individual  months,  and  in  some  whole  years,  the  effluent 
contained  more  than  the  influent. 

The  seasonal  variations  in  the  bacterial  content  of  a 
large  pond  or  lake  follow  a  somewhat  different  course  from 
those  observed  in  a  stream.  Philbrick,  in  the  paper  just 
cited,  gives  the  figures  tabulated  on  following  page  for 
the  Chestnut  Hill  Reservoir  of  the  Metropolitan  Water 
Works  (Boston).  The  averages  are  based  on  weekly 
analyses  covering  the  eleven  years,  1893-1903. 


12 


Elements  of  Water  Bacteriology. 


MONTHLY    VARIATIONS    IN    BACTERIAL    CONTENT    OF 
CHESTNUT    HILL    RESERVOIR,    1893-1903. 


Month 

J. 

F. 

M. 

A. 

M. 

J. 

J. 

A. 

S. 

0. 

N. 

D. 

Bacteria 

per  c.c. 

82 

73 

71 

123 

69 

73 

82 

95 

134 

89 

103 

96 

The  marked  increase  in  April  and  September  is  the 
notable  feature  of  these  analyses;  and  this  is  due  to  the 
effect  of  the  spring  and  fall  overturns  which,  in  the  months 
in  question,  stir  up  the  decomposing  organic  matter  at  the 
bottom  and  distribute  it  through  the  reservoir.  The 
slight,  but  steady,  increase  during  the  warm  months  from 
May  to  August  is  also  probably  significant. 

On  the  whole  it  may  be  said  that  the  bacterial  content 
of  a  lake  or  pond  should  not  be  more  than  one  or  two 
hundred  per  c.c.  and  may  often  be  under  a  hundred.  The 
student  will  find  numerous  analyses  of  natural  waters  in 
Frankland's  classic  work  (Frankland,  1894).  He  notes, 
for  example,  that  the  Lake  of  Lucerne  contained  8  to  51 
bacteria  per  c.c.,  Loch  Katrine  74,  and  the  Loch  of  Lin- 
tralthen  an  average  of  170.  The  water  of  Lake  Cham- 
plain  examined  by  one  of  us  (S.  C.  P.)  in  1896  contained 
on  an  average  82  bacteria  per  c.c.  at  a  point  more  than 
two  miles  out  from  the  city  of  Burlington.  Certain 
surface  water-supplies  near  Boston,  studied  by  Nibecker 
and  one  of  us  (Winslow  and  Nibecker,  1903),  gave  the 
following  results: 


The  Bacteria  in  Natural  Waters. 


City. 

Number  of 
Samples. 

Average 
Number 
of  Bacteria 
per  c.c. 

Wakefield 

7 

fn 

Lynn                        .        

6 

16 

Plymouth.     .        .        

6 

•jf 

Cambridge      

c; 

04 

t 

272 

M^edford.                                  .        .           \ 

t 

<24 

Taunton                           

4 

13 

Peabody              

•7 

141 

In  sea-water,  too,  bacterial  numbers  are  small  as  noted 
by  Russell  at  Naples  (Russell,  1891)  and  Wood's  Hole 
(Russell,  1892),  and  in  salt  as  in  fresh  water  the  amount 
of  bacterial  life  decreases  in  general  as  one  passes  down- 
ward from  the  surface  and  outward  from  the  shore.  Otto 
and  Neumann  (1904)  obtained  the  results  summarized 
below  at  various  points  on  the  high  seas  between  Portugal 
and  Brazil.  Near  the  European  coast,  numbers  were 
much  higher. 

BACTERIA  IN  THE  ATLANTIC  OCEAN. 

(OTTO  AND  NEUMANN,  1904.) 

Bacteria  per  c.  c. 


Nearest  Land. 

I 

)epth  in 

Meters. 

5 

5° 

100 

200 

Canary  Islands         

I2O 

76 

20 

I 

q8 

16 

64 

6 

St.  Paul  Island     

20 

480 

"?4 

4" 

Pernambuco                              .    . 

48 

1  68 

14 

"3 

14  Elements  of  Watet  Bacteriology. 

The  decrease  in  numbers  which  takes  place  when  a 
surface  water  is  stored  in  a  pond  or  reservoir,  indicates 
that  the  forces  which  tend  to  produce  bacterial  self- 
purification  are  important  ones.  It  is  necessary  to  con- 
sider in  somewhat  more  detail  just  what  these  forces  are,  in 
order  to  gauge  their  potency  in  any  particular  instance. 

Chief  of  them  appear  to  be  sedimentation,  the  activity 
of  other  micro-organisms,  light,  temperature,  and  food- 
supply,  and  perhaps  more  obscure  conditions  such  as 
osmotic  pressure. 

The  subsidence  of  bacteria  either  by  virtue  of  their  own 
specific  gravity  or  as  the  result  of  their  attachment  to 
particles  of  suspended  matter  is  unquestionably  partly, 
if  not  largely,  responsible  for  changes  in  the  number  of 
bacteria  in  the  upper  layers  of  water  at  rest  or  in  very 
sluggish  streams.  The  results  of  numerous  investigations 
by  different  workers  seem  to  indicate  that  sedimentation 
of  the  bacteria  themselves  takes  place  slowly,  and  that 
the  difference  in  numbers  between  the  top  layer  and  the 
bottom  layer  of  water  in  tall  jars  in  laboratory  experi- 
ments of  a  few  days'  duration  is  very  slight  or  quite  within 
the  limits  of  experimental  error  (Tiemann  and  Gartner, 
1889).  Different  species  may,  of  course,  be  differently 
affected  (Scheurlen,  1891).  It  must  be  remembered, 
however,  that  in  natural  streams  bacteria  are  to  a  great 
extent  attached  to  larger  solid  particles  upon  which  the 
action  of  gravity  is  more  important.  Spitta  (1903) 
found  that  from  one-fifth  to  one-half  of  the  bacteria  in 


The  Bacteria  in  Natural  Waters.  15 

canal  water  may  be  attached  to  gross  particles,  as 
evidenced  by  their  sedimentation  in  a  few  hours. 
Jordan  (Jordan,  1900)  is  firmly  of  the  opinion  that  in  the 
lower  part  of  the  Illinois  River,  where  there  is  a  fall  of 
but  30  feet  in  225  miles,  the  influences  summed  up  by  the 
term  sedimentation  are  sufficiently  powerful  to  obviate 
the  necessity  for  summoning  another  cause  "to  explain 
the  diminution  in  numbers  of  bacteria,"  and  he  further 
adds:  "It  is  noteworthy  that  all  the  instances  recorded 
in  the  literature  where  a  marked  bacterial  purification 
has  been  observed,  are  precisely  those  where  the  conditions 
have  been  most  favorable  for  sedimentation." 

Little  is  known  as  to  the  share  of  other  organisms  in 
hastening  the  decrease  of  bacteria  in  stored  water. 
Doubtless  predatory  Protozoa  play  some  part  in  the 
process.  Huntemuller  (1905),  after  infecting  water 
containing  Protozoa  with  typhoid  bacilli,  found  the 
Protozoa  crowded  with  bacteria;  and  he  observed  under 
the  microscope  the  actual  ingestion  of  the  living  and 
motile  bacilli.  Korschun  (1907)  and  others  have  ob- 
tained similar  results  and  consider  the  activity  of  Protozoa 
to  be  an  important  factor  in  self-purification. 

Fehrs  (1906)  found  that  typhoid  bacilli  would  live 
for  7  days  in  unsterilized  Gottingen  tap  water,  for  46 
days  in  the  same  water  sterilized,  and  for  13  days  in  water 
inoculated  with  a  culture  of  flagellate  Protozoa  after 
sterilization.  Water  bacteria  were  of  course  added 
with  the  Protozoa. 


1 6  Elements  of  Water  Bacteriology. 

Certain  bacteriologists  have  held  that  the  toxic  waste 
products  of  the  bacteria  themselves  may  render  water 
unfit  for  their  own  development.  Horrocks  (Horrocks, 
1901),  Garr^  (Garre,  1887),  Zagari  (Zagari,  1887),  and 
Freudenreich  (Freudenreich,  1888)  have  shown  that  an 
"antagonism"  exists  when  bacteria  are  grown  in  artificial 
culture  media  such  that  the  substratum  which  has 
supported  the  growth  of  one  form  may  be  rendered  anti- 
septic to  another.  Frost  (1904)  has  exhaustively  studied 
the  phenomenon  of  antagonism  by  exposing  typhoid 
bacilli  in  collodion  sacs  to  the  action  of  certain  soil  and 
water  bacteria  growing  in  broth.  Artificial  culture 
media,  however,  offer  conditions  for  bacterial  development 
which  are  scarcely  paralleled  in  natural  waters.  It  is 
difficult  to  believe  that  under  ordinary  conditions  poisons 
are  produced  of  such  power  as  to  render  a  stream  or 
lake  specifically  toxic  for  any  particular  type  of  bacteria. 
It  appears  indeed  from  the  experiments  of  Jordan, 
Russell  and  Zeit  (1904),  and  Russell  and  Fuller  (1906), 
which  will  shortly  be  referred  to  more  fully,  that  the  life 
of  typhoid  germs  is  shorter  in  water  containing  large 
numbers  of  other  bacteria  than  in  that  of  greater 
purity.  Horrocks  (1899),  too,  found  freshly  isolated 
typhoid  bacilli  alive  in  sterile  sewage  after  sixty  days; 
while  they  disappeared  in  five  days  when  B.  coli  was 
also  present.  These  phenomena  may  be  due,  however, 
to  a  struggle  for  oxygen,  or  for  food,  rather  than  to  the 
assumed  presence  of  highly  toxic  bacterial  products  of 
which  there  is  no  independent  evidence. 


The  Bacteria  in  Natural  Waters.  17 

Temperature  has  a  direct  relation  to  bacterial  life,  and 
the  number  of  parasitic  bacteria  at  least  may  be  quickly 
lessened  by  the  action  of  cold.  Sedgwick  and  one  of 
us  (Sedgwick  and  Winslow,  1902)  have  shown  that  of 
typhoid  or  colon  bacilli  in  ice  or  cool  water,  over  40  per 
cent  will  perish  in  three  hours  and  98  per  cent  and  up- 
wards in  two  weeks.  This  diminution  is  not  of  course 
due  to  cold  alone,  but  at  high  temperature  the  decrease 
is  much  less  marked.  At  Harrisburg,  Pa.  (1907),  an 
actual  increase  of  colon  bacilli  was  observed  in  a  small 
reservoir  during  a  warm  period  when  its  temperature 
approximated  blood  heat. 

Many  investigations  conducted  since  the  pioneer  re- 
searches of  Downes  and  Blunt  (Downes  and  Blunt,  1877) 
have  confirmed  the  results  reported  by  them,  which  showed 
that  direct  sunlight  is  fatal  to  most  bacteria  in  the  vegetative 
state  and  even  to  spores  if  the  exposure  be  sufficiently 
long,  while  diffused  light  is  harmful  in  a  less  degree. 
Opinions  vary  as  to  the  degree  to  which  light  is  active 
in  destroying  the  bacteria  in  natural  waters.  Buchner 
(Buchner,  1893)  found  by  experiment  that  the  bacteri- 
cidal power  of  light  extends  to  a  depth  of  about  three 
meters  before  it  becomes  imperceptible.  On  the  other 
hand,  Procaccini  (Procaccini,  1893)  found  that  when  sun- 
light was  passed  vertically  through  60  cm.  of  drain-water 
the  lower  layers  contained  nearly  as  many  bacteria  after 
three  hours'  treatment  as  before  exposure.  The  middle 
and  upper  portions  showed  a  great  falling  off  in  numbers, 
however. 


1 8  Elements  of  Water  Bacteriology. 

But  few  studies  have  been  made  of  the  effect  of  light 
on  bacteria  in  flowing  water.  Jordan  (Jordan,  1900)  has 
investigated  several  Illinois  streams,  and  arrived  at  the 
conclusion  that  in  moderately  turbid  water,  at  least,  the 
sun's  rays  are  virtually  without  action.  On  the  other 
hand,  Rapp  has  observed  a  considerable  reduction  of  the 
bacteria  in  the  Isar  at  Pullach  after  the  period  of  diurnal 
insolation,  as  shown  by  the  table  on  page  20. 

It  is  unnecessary  to  dwell  in  detail  upon  the  effect  which 
the  lack  of  nutritive  elements  must  exert  upon  intestinal 
bacteria  and  soil  bacteria  in  waters  of  ordinary  purity. 
Comparative  studies  of  culture  media,  to  be  quoted  in  the 
succeeding  chapter,  will  show  how  delicately  the  bacteria 
respond  to  comparatively  slight  changes  in  their  food 
supply.  Wheeler  (1906)  found  that  typhoid  bacilli  would 
persist  in  almost  undiminished  numbers  in  sterilized  water 
from  a  polluted  well,  containing  considerable  organic 
matter  and  kept  in  the  dark  at  20  degrees,  while  in  purer 
water  or  in  the  light  they  died  out  in  from  two  to  six  weeks. 

Whipple  and  Mayer  (1906)  have  called  attention  to 
another  important  factor  in  the  general  problem.  They 
iind  that  the  presence  of  oxygen  is  essential  to  the  per- 
sistence of  typhoid  and  colon  bacilli  in  water,  although 
in  nutrient  media  both  forms  may  thrive  under  anaerobic 
conditions. 


The  Bacteria  in  Natural   Waters. 


EFFECT    OF    OXYGEN    ON    VIABILITY    OF    TYPHOID 

BACILLI   IN    STERILE   TAP  WATER. 

(WHIPPLE  AND   MAYER,  1906.) 


Period  in  Days. 

Tubes  kept  in  Air. 

Tubes  kept  in  Hydrogen. 

Bacteria 

Bacteria 

per  c.c. 

Per  Cent. 

per  c.c. 

Per  Cent. 

0 

600,000 

100.  O 

600,000 

IOO.O 

2 

455>°°° 

76.0 

2,400 

0.4 

4 

190,000 

32.0 

25 

0.004 

8 

120,000 

20.  O 

0 

0.0 

12 

67,000 

II.  0 

o 

o.o 

18 

25,000 

4.2 

0 

0.0 

26 

9>25° 

i-5 

0 

o.o 

33 

2,150 

0.6 

0 

0.0 

40 

132 

0.02 

0 

o.o 

47 

6 

O.  OOI 

o 

0.0 

54 

o 

o.ooo 

0 

0.0 

Various  inorganic  constituents  of  the  medium  un- 
doubtedly exercise  an  important  influence  upon  the  life 
of  bacteria  in  water;  and  the  mutual  interaction  of  the 
different  substances  present  is  a  highly  complex  one. 
Thus  Winslow  and  Lochridge  (1906)  report  that  five  parts 
of  dissociated  hydrogen  per  million  parts  of  tap  water 
(.005  normal  HC1)  is  fatal  to  typhoid  bacilli,  while  ten 
times  as  much  acid  is  required  for  sterilization  when  one 
per  cent  of  peptone  is  present  to  check  the  dissociation  of 
the  hydrogen. 

Although  it  is  hard  to  estimate  the  exact  importance  of 
each  factor,  the  general  phenomena  of  the  self-purification 
of  streams  are  easy  to  comprehend.  A  small  brook 
immediately  after  the  entrance  of  polluting  material  from 
the  surface  of  the  ground  contains  many  bacteria  from  a 
diversity  of  sources.  Gradually  those  organisms  adapted 


20 


Elements  of  Water  Bacteriology. 


EXAMINATIONS    OF    THE    ISAR    AT   PULLACH. 

(RAPP,  1903.) 
(A)    Carried  out  September  26,  1898,  no  rain  having  fallen  for  three  weeks. 


Temperature 

Time  of  the 

Bacteria 

of  the  Water. 

of  the  Air. 

I3.0°C. 

8.8°C. 

7.  30  p.m. 

146 

I2.I°C. 

7.o°C. 

9.30  p.m. 

270 

10.  5°  C. 

6.2°C. 

5.00  a.m. 

37° 

10.  2°  C. 

8.2°C. 

8.00  a.m. 

320 

(B)    Carried  out  November  28,    1898,  no  rain  having  fallen  for  some  time. 


5-5°  C. 

3-o°C. 

6.00  p.m. 

266 

S-5°C. 

2.5°C. 

8.00  p.m. 

402 

5-5°C. 

2.0°C. 

10.00  p.m. 

482 

5-o°C. 

2.0°C. 

3.00  a.m. 

532 

4-5°  C. 

2.5°C. 

7.30  a.m. 

400 

to  life  in  the  earth  or  in  the  bodies  of  plants  and  animals 
die  out,  and  the  forms  for  which  water  furnishes  ideal 
conditions  survive  and  multiply.  It  is  no  single  agent 
which  brings  this  about,  but  that  complex  of  little-under- 
stood conditions  which  we  call  the  environment.  If  any 
one  thing  is  of  prime  importance  it  is  probably  the  food- 
supply,  for  only  certain  bacteria  are  able  to  multiply  in 
the  presence  of  the  small  amount  of  organic  matter  present 
in  ordinary  potable  waters.  As  Jordan  (Jordan,  1900) 
has  said:  "In  the  causes  connected  with  the  insufficiency 
or  unsuitability  of  the  food-supply  is  to  be  found,  I  believe, 
the  main  reason  for  the  bacterial  self-purification  of 
streams." 


The  Bacteria  in  Natural   Waters.  21 

It  is  obvious  that  the  efficiency  of  all  the  agencies  which 
tend  to  decrease  the  number  of  bacteria  in  surface  waters 
will  increase  with  the  prolongation  of  the  period  for  which 
they  act.  Time  is  the  great  measure  of  self-purification. 
The  longer  the  storage  period,  the  greater  the  improve- 
ment.* 

*  The  absolute  time  necessary  to  remove  disease  bacteria  and  render 
a  polluted  water  safe  for  drinking  is  impossible  to  fix  with  any  cer- 
tainty. Food  supply,  light,  temperature  and  the  activity  of  other  living 
forms  vary  widely,  and  in  deposited  material  conditions  are  different 
from  those  which  obtain  in  the  water  itself.  Jordan,  Russell  and  Zeit 
(1904),  in  an  important  series  of  experiments,  added  typhoid  bacilli  to 
the  unsterilized  waters  of  Lake  Michigan,  the  Chicago  River  and  Drain- 
age Canal  and  the  Illinois  River,  in  collodion  sacs  suspended  in  the 
respective  bodies  of  water.  From  the  relatively  pure  Lake  Michigan 
water  the  specific  organisms  could  be  isolated  for  at  least  a  week,  but  in 
the  polluted  waters  of  the  rivers  and  the  Drainage  Canal  they  were  not 
found  after  three  days  except  in  a  single  instance.  Russell  and  Fuller, 
(1906)  confirmed  these  general  results,  finding  that  typhoid  bacilli 
would  live  for  ten  days  in  the  unsterilized  water  of  Lake  Mendota  while 
they  could  isolate  them  only  after  five  days  when  immersed  in  sewage. 
Other  observers  record  much  greater  viability  for  the  typhoid  bacillus. 
Savage  (1905)  added  a  heavy  dose  of  the  organism  to  unsterilized  tidal 
mud  and  found  it  living  after  five  weeks.  Hoffmann  (1905),  after 
inoculating  a  large  aquarium  with  a  rich  typhoid  culture,  was  able  to 
isolate  the  germ  from  the  water  after  four  weeks  and  from  the  mud  at 
the  bottom  after  two  months.  Konrddi  (1904)  reports  the  persistence 
of  typhoid  bacilli  in  unsterilized  tap  water  for  over  a  year. 

Under  certain  conditions  it  is  even  possible  for  intestinal  bacteria  to 
multiply  in  water.  At  Harrisburg,  Pa.,  a  series  of  B.  coli  examina- 
tions made  in  the  midsummer  of  1906  showed  positive  results  in  7  per 
cent  of  the -samples  of  water  entering  the  storage  reservoir  and  in  27 
per  cent  of  the  samples  leaving  it.  The  storage  period  in  this  case  was 
about  two  days  and  the  temperature  of  the  water  in  the  reservoir  nearly 
at  blood  heat  (Harrisburg,  1907).  It  is  improbable  that  true  patho- 
genic germs  like  the  typhoid  bacillus  could  increase  even  under  such 
exceptional  conditions. 


22  Elements  of  Water  Bacteriology. 

• 

In  general  we  have  seen  that  surface-waters  tend 
continually  to  decrease  in  bacterial  content  after  their 
first  period  of  contact  with  the  humus  layer  of  the  soil. 
In  that  other  portion  of  the  meteoric  water  which  pene- 
trates below  the  surface  of  the  earth  to  join  the  reservoir 
of  ground-water,  later  to  reappear  as  the  flow  of  springs 
and  wells,  this  diminution  is  still  more  marked  since  the 
filtering  action  of  the  earth  removes  not  only  most  of  the 
bacteria  but  much  of  their  food  material  as  well.  The 
numbers  of  bacteria  in  the  soil  itself  decrease  rapidly  as 
one  passes  downward.  Kabrhel  (1906)  found  several 
million  per  c.c.  in  surface  samples  of  woodland  soil,  a  few 
thousands  or  tens  of  thousands  half  a  meter  below  and 
usually  only  hundreds  in  centimeter  samples  collected  at 
depths  greater  than  a  meter.  Many  observers  formerly 
believed  that  all  ground-waters  were  nearly  free  from 
bacteria,  because  often  no  colonies  appeared  on  plates 
counted  after  the  ordinary  short  periods  of  time.  If, 
however,  a  longer  period  of  incubation  be  adopted  consid- 
erable numbers  may  be  obtained. 

For  convenience  we  may  divide  ground-waters  into 
three  groups,  namely:  shallow  open  wells,  springs,  and 
"tubular"  (driven)  or  deep  wells.  This  division  is  impor- 
tant because  ordinary  shallow  wells  form  a  group  by 
themselves  in  respect  to  the  possibility  of  aerial  and  surface 
contamination,  their  water  often  being  fairly  rich  in  bac- 
terial life.  Egger  (Wolffhiigel,  1886)  examined  60  wells 
in  Mainz  and  found  that  17  of  them  contained  over  200 


The  Bacteria  in  Natural  Waters.  23 

bacteria  to  the  cubic  centimeter.  Maschek  (Maschek, 
1887)  found  36  wells  out  of  48  examined  in  Leitmeritz 
which  had  a  bacterial  content  of  over  500  per  c.c.  Fischer 
(Horrocks,  1901)  reported  120  wells  in  Kiel  which  gave 
over  500  bacteria  per  c.c.  and  only  51  with  less  than  that 
number. 

In  the  ordinary  standard  48-hour  period  very  few  bac- 
teria develop  from  normal  spring-waters.  Thus  in  an 
examination  of  spring-waters  made  by  the  Massachusetts 
State  Board  of  Health  in  1900  (Massachusetts  State 
Board  of  Health,  1901),  of  37  springs  which  were  prac- 
tically unpolluted  and  had  less  than  o.io  parts  per  100,000 
excess  of  chlorine  over  the  normal,  54  samples  were  exam- 
ined and  gave  an  average  of  41  bacteria  per  c.c.  Only 
6  samples  showed  figures  over  50. 

It  now  remains  to  consider  the  other  great  division  of 
ground-waters,  namely,  deep,  "driven,"  or  "tubular" 
wells,  which,  if  carefully  constructed,  should  ordinarily  be 
free  from  all  surface-water  contamination,  and  should 
show  low  bacterial  counts.  The  results  tabulated  below 
obtained  by  Houston  in  the  examination  of  a  series  of  deep 
wells  of  high  quality  at  Tunbridge  Wells  are  fairly  typical. 

BACTERIAL  CONTENT  OF  DEEP  WELL  WATERS. 

(HOUSTON,  1903.) 

Bacteria  per  c.c. 


36 

6 

9 

4 

i 

16 

17 

4 

3 

12 

2 

4 

10 

5 

2 

24 


Elements  of  Water  Bacteriology. 


Fifteen  driven  wells  in  the  neighborhood  of  Boston,  ex- 
amined in  1903,  showed  at  the  end  of  48  hours  an  average 
of  only  18  colonies  per  c.c.;  and  the  results  of  certain 
examinations  of  other  wells  and  springs,  recently  made 
by  the  authors,  are  given  in  the  table  below. 

BACTERIA  IN  DEEP  WELL  AND  SPRING  WATERS. 


Town. 

Bacteria 
per  c.c. 

Town. 

Bacteria 
per  c.c. 

Worcester,  Mass.  . 
Waltham,  Mass.  .  . 
Newport  R  I 

10 

3 

7" 

Saranac  Lake,  N.Y. 
Ellenville,  N.  Y.  . 
Hyde  Park  Mass 

II 
o 

12 

It  is  plain  that  water  absolutely  free  from  bacteria  is 
not  ordinarily  obtained  from  any  source.  In  deep  wells, 
however,  their  number  is  small;  and  the  peculiar  character 
of  the  organisms  present  is  manifested  in  many  cases  by 

• 

the  slow  development  at  room  temperature  (frequently  no 
growth  until  the  third  day),  the  entire  absence  of  liquefying 
colonies,  and  the  abundance  of  chromogenic  species. 


CHAPTER  II. 

THE  QUANTITATIVE  BACTERIOLOGICAL  EXAMINATION 
OF  WATER. 

THE  customary  methods  for  determining  the  number  of 
bacteria  in  water  do  not  reveal  the  total  bacterial  content, 
but  only  a  very  small  fraction  of  it,  as  becomes  apparent 
when  we  consider  the  large  number  of  organisms,  nitrify- 
ing bacteria,  cellulose-fermenting  bacteria,  strict  anae- 
robes, etc.,  which  refuse  to  grow,  or  grow  only  very  slowly 
in  ordinary  culture  media,  and  which,  therefore,  escape 
detection.  On  the  one  hand,  certain  obligate  parasites 
cannot  thrive  in  the  absence  of  the  rich  fluids  of  the 
animal  body;  on  the  other  hand,  the  prototrophic  bacteria 
adapted  to  the  task  of  wrenching  energy  from  nitrates 
and  ammonium  compounds  are  unable  to  develop  in  the 
presence  of  so  much  organic  matter.  Winslow  (1905), 
in  the  examination  of  sewage  and  sewage  effluents,  found 
20-70  times  as  many  bacteria  by  microscopic  enumeration 
as  by  the  gelatin  plate  count.  Certain  special  media 
enable  us  to  obtain  much  larger  counts  than  those  yielded 
by  the  ordinary  gelatin  method.  The  Nahrstoff  Heyden 
agar,  for  example,  has  been  strongly  advocated  by  Hesse 
(Hesse  and  Niedner,  1898)  and  other  German  bacteriol- 
ogists upon  this  ground.  In  this  country  Gage  and  Phelps 
(Gage  and  Phelps,  1902)  showed  that  the  numbers  obtained 

25 


26  Elements  of  Water  Bacteriology* 

by  the  ordinary  procedure  were  only  from  5  to  50  per  cent 
of  those  obtained  by  the  use  of  Heyden's  Nahrstoff  agar. 
For  practical  sanitary  purposes,  however,  our  methods  are 
fairly  satisfactory.  Within  limits,  it  is  of  no  great  impor- 
tance that  one  method  allows  the  growth  of  more  bacteria 
than  another.  When  we  are  using  the  quantitative  analy- 
sis as  a  measure  of  sewage  pollution  the  essential  thing  is 
that  the  section  of  the  total  bacterial  flora  which  we  obtain 
should  be  thoroughly  representative  of  that  portion  of  it  in 
which  we  are  most  interested  —  the  group  of  the  quickly 
growing,  rich-food-loving  sewage  forms.  In  this  respect 
meat-gelatin-peptone  appears  to  be  unrivalled;  and  it  is 
in  this  respect  that  such  media  as  Nahrstoff  agar  fail. 
Miiller  (1900)  showed  that  the  larger  counts  obtained 
by  plating  on  the  Nahrstoff  medium  are  due  to  the  fact 
that  it  specially  favors  the  more  prototrophic  forms,  among 
the  water  bacteria  themselves.  Intestinal  organisms  and 
even  the  ordinary  putrefactive  germs,  when  plated  in  pure 
culture,  show  no  higher  counts  on  Nahrstoff  agar  than  on 
gelatin.  Gage  and  Adams  (1904)  found  by  plating  pure 
cultures  of  the  common  laboratory  bacteria,  saprophytes, 
and  parasites,  that  Nahrstoff  counts  were  actually  lower 
than  those  obtained  by  the  use  of  gelatin.  When  sewage 
and  highly  polluted  waters  are  examined,  counts  are 
slightly  higher  on  Nahrstoff  media,  while  with  purer 
waters  the  Nahrstoff  numbers  are  far  in  excess  of  those 
obtained  with  gelatin.  Winslow  (1905)  found  the  ratio 
of  Nahrstoff  agar  to  gelatin  count  to  be  1.7  to  i.o  for 


Quantitative  Bacteriological  Examination.        2/ 


sewage,  and  4.8  to  i.o  for  sand  filter  effluent.  With  waters 
of  still  better  quality  the  ratio  goes  higher,  reaching  a 
maximum  when  the  bacteria  which  increase  and  multiply 
in  pure  water  are  most  abundant.  Miiller  (1900)  found,  for 
example,  that  water  which  normally  showed  six  times  as 
many  bacteria  on  Nahrstoff  agar  as  on  gelatin  might  give 
a  Nahrstoff-gelatin  ratio  of  20-30  after  it  had  been  stand- 
ing for  some  time  in  the  supply  pipes.  The  table  below, 
taken  from  the  valuable  paper  by  Gage  and  Phelps  (1902), 
shows  strikingly  the  different  Nahrstoff-gelatin  ratios  for 
waters  of  various  grades  of  purity. 

TABLE   SHOWING   PERCENTAGES   OF  BACTERIA  DEVEL- 
OPING    ON     REGULAR     AGAR    AND    ON    NAHRSTOFF 
AGAR  FOR    DIFFERENT    CLASSES    OF   WATERS. 
(GAGE  AND  PHELPS,  1902). 
REGULAR    AGAR. 


Days'  Count. 

Class  of  Water. 

2 

3 

4 

5 

6 

7 

8 

Ground-  water  .    . 

O 

5 

6 

6 

6 

6 

6 

Filtered  water  .    . 

6 

7 

7 

7 

7 

7 

7 

Merrimac  River  . 

6 

7 

7 

8 

8 

9 

9 

Filtered  sewage    . 

14 

*7 

18 

iQ 

iQ 

19 

19 

Sewage  

34 

44 

46 

46 

'   46 

46 

46 

NAHRSTOFF    AGAR. 


Ground-water  . 

6 

43 

78 

88 

93 

IOO 

IOO 

Filtered  water  . 

37 

69 

80 

92 

98 

IOO 

IOO 

Merrimac  River 

29 

78 

93 

97 

97 

99 

IOO 

Filtered  sewage 

26 

65 

93 

95 

97 

99 

IOO 

Sewage  .... 

39 

75 

95 

IOO 

IOO 

IOO 

IOO 

28  Elements  of  Water  Bacteriology. 

It  is  obvious  from  all  these  facts  that  the  effect  of  using 
the  Nahrstoff  medium  is  to  increase  disproportionately 
the  bacterial  counts  obtained  from  purer  waters  and 
thus  to  diminish  the  difference  in  bacterial  content  be- 
tween normal  and  contaminated  sources.  The  ordinary 
gelatin  medium,  on  the  other  hand,  is  adapted  to  the 
growth  of  intestinal  and  putrefactive  forms  and,  therefore, 
serves  best  the  prime  object  of  bacteriological  water  ex- 
amination. 

The  'first  requisite  in  a  procedure  for  water  analysis 
is,  that  it  should  be  adapted  to  the  end  in  view,  the 
differentiation  of  pure  and  contaminated  waters.  The 
second  and  equally  important  requirement  is,  that  the 
procedure  should  be  a  standard  one,  so  that  results 
obtained  at  different  times  and  by  different  observers 
may  be  comparable.  In  this  respect  the  work  of 
G.  W.  Fuller,  G.  C.  Whipple,  and  other  members  of  the 
Committee  on  Standard  Methods  of  the  American  Public 
Health  Association  has  placed  the  art  of  quantitative 
water  analysis  in  this  country  in  a  very  satisfactory  state 
by  contrast  with  the  varying  practices  which  prevail  in 
England  and  Germany.  The  first  report  on  this  question 
was  made  in  1897  (Committee  of  Bacteriologists,  1898). 
A  permanent  Committee  on  Standard  Methods  was  then 
formed  which  reported  in  1901  (Fuller,  1902),  and 
again  in  1904  (Committee  on  Standard  Methods  of 
Water  Analysis,  1905),  recommending  in  considerable 
detail  a  standard  routine  procedure  for  the  quantitative 


Quantitative  Bacteriological  Examination.        29 

and  qualitative  bacteriological  examination  of  water  for 
sanitary  purposes.  These  reports  have  had  a  far-reaching 
effect  in  simplifying  and  unifying  the  methods  of  water 
analysis.  Similar  results  may  be  expected  from  the  work 
of  the  English  Committee  appointed  to  consider  the 
Standardization  of  Methods  for  the  Bacterioscopic  Exam- 
ination of  Water  which  reported  in  1904,  although  this 
committee  unfortunately  did  not  consider  the  process  of 
media  making  in  great  detail.  The  last  report  of  the 
American  Committee  on  Standard  Methods  (1905)  will 
be  adhered  to  in  this  and  succeeding  chapters  unless 
otherwise  specifically  stated;  and  that  portion  of  its 
report  which  deals  with  methods  of  making  media  will 
be  found  in  full  in  the  Appendix. 

The  procedure  for  the  quantitative  determination  of 
bacteria  in  water  consists,  in  brief,  in  mixing  a  definite 
amount  of  a  suitably  collected  specimen  of  the  water  with 
a  sterile  solidifiable  culture  medium  and  allowing  it  to 
develop  for  a  sufficiently  long  time  to  permit  reproduction 
of  the  bacteria  and  the  formation  of  visible  colonies  which 
may  be  counted.  The  process  is  divided  naturally 
into  four  stages  —  sampling,  plating,  incubating,  and 
counting. 

Sampling.  —  All  samples  of  water  for  bacteriological 
examination  should  be  collected  in  clean,  sterile  bottles 
with  wide  mouths  and  glass  stoppers,  preferably  of  the  flat 
mushroom  type.  It  is  desirable  that  these  bottles  should 
have  a  capacity  of  at  least  100  c.c. 


3O  Elements  of  Water  Bacteriology. 

They  should  be  cleaned  thoroughly  before  using,  by 
treatment  with  sulphuric  acid  and  potassium  bichromate 
or  with  alkaline  permanganate  of  potash,  followed  by 
sulphuric  acid,  dried  by  draining,  and  sterilized  by  dry 
heat  at  160°  C.  for  at  least  one  hour,  or  by  steam  at  115- 
120  degrees  for  fifteen  minutes.  If  not  to  be  used  immedi- 
ately the  neck  and  stopper  should  be  protected  against 
dust  or  other  contamination  by  wrapping  with  lead-foil. 
For  transportation  the  bottle  should  be  enclosed  in  a 
suitable  case  or  box. 

The  greatest  care  must  be  taken  that  the  fingers  do  not 
touch  the  inside  of  the  neck  of  the  bottle  or  the  cone  of 
the  stopper,  as  the  water  thereby  would  become  seriously 
contaminated  and  rendered  unfit  for  examination.  It  is 
well  known  that  bacteria  are  found  abundantly  upon  the 
skin,  and  Winslow  (Winslow,  1903)  has  shown  that  even 
B.  coli  is  present  upon  the  hands  in  a  considerable  number 
of  cases. 

In  order  to  obtain  a  fair  sample,  great  precautions  must 
be  taken,  and  these  will  vary  with  the  different  classes  of 
waters  to  be  examined  and  with  local  conditions.  If  a 
sample  is  to  be  taken  from  a  tap,  the  water  should  be 
allowed  to  flow  at  least  five  minutes  (if  from  a  tap  in  regu- 
lar use)  or  for  a  longer  period  in  case  the  water  has  been 
standing  in  the  house  service  system.  In  the  small  pipes, 
changes  in  bacterial  content  are  liable  to  occur,  certain 
species  dying  and  others  multiplying. 

If  a  sample  is  to  be  taken  from  a  pump  similar  pre- 


Quantitative  Bacteriological  Examination.        31 

cautions  are  necessary.  The  pump  should  be  in  con- 
tinuous operation  for  five  minutes  at  least,  and  preferably 
for  half  an  hour  before  the  sample  is  taken,  in  order  to 
avoid  excessively  high  numbers  due  to  the  growth  of 
bacteria  within  the  well  and  pump,  the  bacterial  condition 
of  the  water  as  it  passes  through  the  ground  being  what 
we  wish  to  determine.  Thus  Heraeus  (Heraeus,  1886),  in 
a  well-water  which  had  been  but  little  used  during  the 
preceding  thirty-six  hours,  found  5000  organisms  per  c.c. ; 
when  the  well  was  emptied  by  continuous  pumping,  a 
second  sample,  after  an  interval  of  half  an  hour,  gave 
only  35.  Maschek  (Tiemann  and  Gartner,  1889)  obtained 
similar  results  'shown  in  the  following  table: 

EFFECT    OF   PUMPING    ON    THE    BACTERIAL   CONTENT 
OF  WELL-WATER. 

Well-water  after  continuous  pumping  for  fifteen  minutes     ....  458 

after  continuous  pumping  for  many  hours 140 

later 68 

'           after  continuous  pumping  for  fifteen  minutes     ....  578 

after  continuous  pumping  for  many  hours 179 

later 73 

After  a  proper  interval  of  pumping  the  sample  of  a  well- 
water  may  be  collected  from  the  pet-cock  of  the  pump  or 
from  a  near-by  tap.  With  a  hand-pump,  such  as  is  found 
in  domestic  shallow  wells,  the  water  is,  of  course,  pumped 
directly  into  the  sample  bottle.  The  difficulties  in  securing 
an  average  sample  from  this  latter  source  are  often  great, 
since  if  the  flooring  about  the  pump  is  not  tight,  as  is 


32  Elements  of  Water  Bacteriology. 

usually  the  case,  continued  pumping  may  wash  in  an 
unusual  amount  of  surface  pollution. 

In  sampling  surface-waters,  the  greatest  precautions 
must  be  observed  to  prevent  contamination  from  the 
fingers.  In  still  waters  the  fairest  sample  is  one  taken 
from  several  inches  down,  as  the  surface  itself  is  likely  to 
have  dust  particles  floating  upon  it.  The  method  most 
frequently  recommended  is  to  plunge  the  bottle  beneath 
the  surface  to  a  depth  of  a  foot  or  so,  then  removing  the 
stopper  and  allowing  the  bottle  to  fill. 

Another  method  which  is  comparatively  free  from 
objection,  and  which  has  been  employed  by  the  writers,  is 
to  remove  the  stopper  first  and  then,  holding  the  bottle  by 
the  base,  plunge  it  mouth  downward  into  the  water,  turn- 
ing it  at  the  desired  depth  so  as  to  replace  the  enclosed 
air  by  the  water.  Whenever  any  current  exists,  the  mouth 
of  the  bottle  should  be  directed  against  it  in  order  to 
carry  away  any  bacteria  from  the  fingers.  If  there  is 
no  current,  a  similar  effect  can  be  produced  by  turning 
the  bottle  under  water  and  giving  it  a  quick  forward 
motion.  In  rapidly  flowing  streams  it  is  only  necessary 
to  hold  the  bottle  at  the  surface  with  the  mouth  pointed 
up-stream. 

For  taking  samples  of  water  at  greater  depths,  a  num- 
ber of  devices  have  been  employed,  all  of  which  are 
fairly  satisfactory.  The  essentials  are,  first,  a  weight 
to  carry  the  bottle  down  to  the  desired  depth,  and,  second, 
some  method  of  removing  the  stopper  when  that  depth 


Quantitative  Bacteriological  Examination.          33 

is  reached.  The  student  will  find  one  good  form  of 
apparatus  described  in  Abbott's  "Principles  of  Bacteri- 
ology" (Abbott,  1899);  an  admirable  one  was  devised 
by  Hill  and  Ellms  (Hill  and  Ellms,  1898);  and  Thresh 
(1904)  figures  an  ingenious  device  for  the  same  purpose. 
Miquel  and  Cambier  (Miquel  and  Cambier,  1902)  and 
other  authors  recommend  the  use  of  a  sealed  glass  bulb 
with  a  capillary  tube  which  can  be  broken  off  at  the 
desired  moment. 

As  soon  as  a  sample  of  water  is  collected,  its  conditions 
of  equilibrium  are  upset  and  a  change  in  the  bacterial 
content  begins.  Even  in  the  purest  spring-waters,  which 
contain  but  few  bacteria  when  collected,  and  in  which 
the  amount  of  organic  matter  is  infinitesimal,  enormous 
numbers  may  be  found  after  storage  under  laboratory 
conditions  for  a  few  days  or  even  a  few  hours.  In  some 
cases  the  rise  in  numbers  is  gradual,  in  others  very  rapid. 
The  Franklands  (Frankland,  1894)  record  the  case  of  a 
deep-well  water  in  which  the  bacteria  increased  from 
7  to  495,000  in  three  days.  Miquel  (Miquel,  1891)  from 
his  researches,  arrived  at  the  conclusion  that  in  surface- 
waters  the  rise  is  less  rapid  than  in  waters  from  deep 
wells  or  springs,  and  that  in  the  latter  case  the  decrease, 
after  reaching  a  maximum,  is  likewise  rapid  and  steady. 
Just  how  far  protection  from  light,  increase  in  tempera- 
ture, and  a  destruction  of  higher  micro-organisms  is 
responsible  for  the  increase,  and  to  what  extent  an  exhaus- 
tion of  food-supply  or  the  formation  of  toxic  waste 


34 


Elements  of  Water  Bacteriology. 


products   causes   the   succeeding  decrease,   we   are  not 
aware;  but  the  facts  are  well  established. 

Whipple  has  exhaustively  studied  the  details  of  this  mul- 
tiplication of  bacteria  in  stored  waters,  and  has  shown 
in  the  table  given  below  that  there  is  first  a  slight  reduc- 
tion in  the  number  present,  lasting  perhaps  for  six  hours, 
followed  by  the  great  increase  noted  by  earlier  observers. 
It  is  probable  that  there  is  a  constant  increase  of  the 
typical  water  bacilli,  overbalanced  at  first  by  a  reduction 
in  other  forms,  for  which  the  environment  is  unsuitable. 


BACTERIAL  CHANGES  IN  WATER  DURING  STORAGE. 
(WHIPPLE,  1901.) 


Temp. 

Number  of  Bacteria  per  c.c. 

Initial 

of  Incu- 

Sample. 

Temper- 

bation 

ature. 

of 

Initial. 

After 

After 

After 

After 

Sample. 

3  Hours. 

6  Hours. 

£4  Hours. 

48  Hours. 

C. 

C. 

A 

7.6° 

17.0° 

260 

215 

230 

900 

27,000 

B 

7.6° 

I7.o° 

260 

245 

255 

720 

10,850 

C 

7-6° 

12-5° 

260 

270 

231 

600 

2,790 

D 

7-6° 

12-5° 

260 

270 

245 

710 

1,  800 

E 

7-6° 

2.4° 

260 

243 

210 

675 

1,980 

F 

7.6° 

2.4° 

260 

235 

270 

56o 

1,980 

G 

11.0° 

12.8° 

77 

55 

58 

101 

10,250 

H 

11.0° 

12.8° 

77 

53 

74 

87 

2,175 

I 

11.0° 

23.6° 

77 

51 

52 

11,000 

41,400 

J 

6-7° 

20.0° 

43° 

375 

245 

... 

38S,oool 

K 

6-7° 

20.0° 

43° 

345 

405 

... 

750,000* 

L 

23.2° 

23.00 

Sio 

34o 

230 

8,000 

20,000 

M 

23.2° 

2-5° 

S25 

300 

220 

380 

2,200 

1  0.0005  Per  cent  peptone  added  to  the  water. 


Quantitative  Bacteriological  Examination.        35 


EFFECT  OF  SIZE   OF  VESSEL  UPON   THE  MULTIPLICA- 
TION  OF  WATER  BACTERIA  DURING   STORAGE. 

(WHIPPLE,  1901.) 


Temp. 

Number  of  Bacteria  per  c.c. 

Sample 

Bottle. 

of  In- 

cuba- 

Ini- 

After 

After 

After 

After 

After 

tion. 

tial.1 

3  hrs. 

6  hrs. 

12  hrs. 

24  hrs. 

48  hrs. 

C. 

A 

i  -gallon 

13° 

77 

63 

65 

47 

42 

175 

B 

2  -quart 

13° 

77 

59 

63 

60 

45 

690 

C 

i  -quart 

13° 

77 

63 

63 

47 

46 

325 

D 

i  -pint 

13° 

77 

57 

61 

36 

38 

630 

E 

2  -ounce 

13° 

77 

55 

58 

47 

101 

10,250 

F 

i  -gallon 

24° 

77 

81 

97 

275 

290 

300 

G 

2  -quart 

24° 

77 

92 

59 

62 

180 

250 

H 

i  -quart 

24° 

77 

84 

77 

46 

340 

900 

I 

i  -pint 

24° 

77 

5i 

46 

100 

2,950 

7,020 

J 

2-ounce* 

24° 

77 

5i 

52 

145 

1  1  ,000 

41,400 

1  Average  of  five  plates. 

Wolffhiigel  and  Riedel  (Wolffhiigel  and  Riedel,  1886) 
noted  the  dependence  of  this  multiplication  on  the  air- 
supply,  vessels  closed  with  rubber  stoppers  showing 
lower  numbers  than  those  plugged  with  cotton.  Simi- 
larly, Whipple  found  that  the  multiplication  of  bacteria 
was  much  greater  when  bottles  were  only  half  full  than 
when  they  were  filled  completely;  and  also,  as  shown 
in  the  above  table  that  the  size  of  the  bottle  markedly 
influenced  the  growth. 

An  important  series  of  investigations  by  Kohn  (1906) 
suggests  that  this  phenomenon  of  multiplication  during 
storage  may  be  due  in  part  to  -the  solution  of  certain 
constituents  of  glass  which  favor  bacterial  life,  since  the 


36  Elements  of  Water  Bacteriology. 

increase  is  notably  greater  in  bottles  of  the  more  soluble 
glasses. 

Whipple's  tables,  quoted  above,  show  that  the  multi- 
plication during  storage  was  greater  at  a  higher  temper- 
ature; and  this  is  a  well  recognized  general  rule.  In 
order  to  obviate  the  abnormal  results  of  storage  increase 
it  is  therefore  obvious  that  samples  must  be  examined 
shortly  after  collection,  and  that  they  must  be  kept  cool 
during  their  necessary  storage.  If  fairly  pure  waters 
are  placed  upon  ice  and  kept  between  o  degrees  and  10 
degrees,  they  will  show  no  material  increase  in  twelve 
hours.  With  polluted  water,  however,  another  danger 
is  here  introduced.  Samples  of  such  water  when  packed 
in  ice  show  a  marked  decrease  due.  to  the  large  number 
of  sensitive  intestinal  bacteria  present.  Jordan  (Jordan, 
1900)  found  that  three  samples  of  river- water  packed  in 
ice  for  forty-eight  hours  fell  off  from  535,000  to  54,500; 
from  412,000  to  50,500,  and  from  329,000  to  73,0x^0, 
respectively.  It  is,  therefore,  important  that  even  iced 
samples  should  not  be  kept  too  long;  and  it  is  desirable 
to  adhere  strictly  to  the  recommendations  of  the  Standard 
Methods  Committee  that  the  interval  between  sampling 
and  examination  should  not  exceed  twelve  hours  in  the 
case  of  relatively  pure  waters,  six  hours  in  the  case  of 
relatively  impure  waters,  and  one  hour  in  the  case 
of  sewage. 

Plating.  —  The  bottle  containing  the  sample  of  water 
is  first  shaken  at  least  twenty-five  times  in  order  to  get  an 


Quantitative  Bacteriological  Examination.         37 

equal  distribution  of  the  bacteria.  If  the  number  of 
bacteria  present  is  probably  not  greater  than  200,  i  c.c.  is 
then  withdrawn  with  a  sterile  i  c.c.  pipette  and  delivered 
into  a  sterile  Petri  dish  of  10  cm.  diameter.  To  this  is 
added  5  c.c.  of  standard  10  per  cent  gelatin  at  a  tempera- 
ture of  about  30°  C.  or  standard  agar  (7  c.c.)  at  4o°-42°  C. 
Should  the  number  of  bacteria  per  c.c.  probably  exceed 
200,  dilution  is  necessary.  This  is  best  accomplished  by 
adding  i  c.c.  of  the  water  in  question  to  9,  99  or  999, 
etc.,  c.c.  of  sterile  tap  water  according  to  the  amount  of 
dilution  required.  The  diluted  sample  is  then  shaken 
thoroughly  and  i  c.c.  taken  for  enumeration.  In  order 
to  determine  the  number  of  bacteria  originally  present 
it  is  only  necessary  to  multiply  by  the  factor  10,  100,  or 
1000,  etc. 

When  a  sample  of  water  from  an  unknown  source  is  to 
be  examined  it  is  generally  desirable  to  make  two  check 
plates  at  each  of  the  above  dilutions,  selecting  those  dilu- 
tions which  give  nearest  to  200  colonies  on  the  plates  after 
incubation  as  the  ones  on  which  to  rely  for  the  count. 
A  much  smaller  number  will  not  give  average  figures, 
and  if  more  than  200  colonies  are  present  on  a  plate  many 
bacteria  will  be  checked  by  the  waste  products  of  those 
which  first  develop  and  the  count  obtained  will  be  too  low. 
After  the  addition  of  the  diluted  sample  and  the  nutrient 
medium,  their  thorough  mixture  in  an  even  layer  on  the 
bottom  of  the  plate  is  obtained  by  careful  tipping  and 
rotation. 


38  Elements  of  Water  Bacteriology. 

It  was  formerly  customary  to  mix  the  water  with  the 
gelatin  in  the  tube  before  pouring  into  the  plate,  but  this 
method  is  objectionable  because  there  is  always  a  resir 
duum  of  medium  remaining  in  the  tube  which  will  retain 
varying  numbers  of  bacteria  and  thus  interfere  with  the 
accuracy  of  the  count.  Before  pouring  the  medium  into 
the  plate  the  mouth  of  the  tube  should  be  flamed  to 
remove  any  possibility  of  contamination. 

The  exact  composition  of  the  medium  is,  of  course,  of 
prime  importance  in  controlling  the  number  of  bacteria 
which  will  develop.  The  figures  previously  cited  in  con- 
'nection  with  the  discussion  of  Hesse's  Nahrstoff  agar 
show  how  bacterial  counts  may  vary  with  media  of  widely 
different  composition.  The  table  on  page  39,  quoted  from 
Gage  and  Phelps  (1902),  shows  the  considerable  dif- 
ferences which  may  be  due  to  the  presence  or  absence  of 
meat  infusion,  peptone,  etc.,  in  media  of  generally  similar 
character  (compare  the  figures  for  plain  gelatin,  pepton 
gelatin,  and  meat  gelatin).  Much  slighter  variations 
than  this,  however,  are  significant.  The  reaction  of  the 
medium  was  found  as  early  as  1891  to  be  important,  for 
Reinsch  (Reinsch,  1891)  showed  in  that  year  that  the 
addition  of  one  one-hundredth  of  a  gram  of  sodium  car- 
bonate to  the  liter  increased  sixfold  the  number  of  bac- 
teria developing.  Fuller  (Fuller,  1895)  and  Sedgwick 
and  one  of  us  (Sedgwick  and  Prescott,  1895),  working 
independently,  established  the  fact  that  an  optimum 
reaction  existed  for  most  water  bacteria,  and  that  a 


Quantitative  Bacteriological  Examination.          39 


deviation  either  way  decreased  the  number  of  colonies 
developing. 

TABLE  SHOWING  PERCENTAGES  OF  BACTERIA  DEVEL- 
OPING  ON   MEDIA   OF   DIFFERENT   COMPOSITIONS. 

(GAGE  AND  PHELPS,  1902.) 


Medium. 

Days'  Count. 

2 

3 

4 

5 

6 

7 

8 

9 

Nahrstoff  agar      
Nahrstoff  pepton  agar     .    . 
Pepton  agar 

19 
10 
II 

8 
8 

6 

7 

12 

1 

I 

5 

60 

22 

16 
13 

10 

9 
10 

7 
19 

12 
10 

6 
6 

78 
26 

22 

16 

i3 
ii 
ii 
8 

24 
18 
ii 

12 

9 

85 
28 

23 
17 
14 
ii 
ii 
8 
26 
20 

12 

13 
II 

95 
3° 
24 
17 
i4 
ii 
ii 
10 
26 

20 
13 
13 
13 

99 
3° 
24 

17 
14 
ii 
ii 

10 

26 

20 
13 
13 
13 

99 
3° 
24 
17 
14 
ii 
ii 
10 
26 

20 
13 
13 
13 

IOO 

3° 
24 
17 
14 
ii 
ii 
10 
26 
20 
13 
13 
13 

Meat  agar     ..'..... 
Plain  agar     
Regular  agar      *  . 

Nahrstoff  glycerin  agar  .    . 
Nahrstoff  meat  agar    .    .    . 
Meat  gelatin     
Pepton  gelatin 

Standard  gelatin  
Plain  gelatin 

Nahrstoff  gelatin      .... 

Whipple  (Whipple,  1902)  has  shown  that  not  only  the 
particular  kind  of  gelatin  used  but  its  exact  physical  con- 
dition as  affected  by  sterilization  and  other  previous 
treatment  will  materially  affect  the  results  obtained. 
Gage  and  Adams  (1904)  found  marked  differences  in 
counts  as  the  result  of  the  use  of  the  two  best  known  com- 
mercial peptones.  A  long  series  of  waters  plated  on  agar 
made  up  with  Merck's  and  Witte's  peptones,  respectively, 
showed  the  following  average  relative  results: 


4O  Elements  of  Water  Bacteriology. 

AVERAGE  RELATIVE  NUMBER  OF  BACTERIA  ON  PEPTON 
AGAR  WITH  DIFFERENT  PEPTONS. 
(GAGE  AND  ADAMS,  1904.) 


Days 

2 

6 

8 

IO 

12 

Merck's  

•?•? 

r  T 

67 

80 

08 

Witte's     

& 

?•? 

IOO 

IOO 

IOO 

IOO 

The  same  authors  showed  that  the  composition  of  the 
water  used  exercised  a  marked  selective  action  upon  the 
development  of  bacteria.  Agar  made  up  with  sewage 
permitted  a  maximum  growth  of  sewage  bacteria  and 
showed  no  colonies  when  inoculated  with  filtered  city 
water.  On  the  other  hand  agar  made  up  with  city 
water  showed  100  per  cent  of  the  bacteria  present  in 
city  water  and  river  water,  three-quarters  of  those  present 
in  sewage  and  less  than  half  of  those  present  in  sewage 
effluents. 

Hesse  (1904)  found  that  the  number  of  bacteria 
developing  on  Nahrstoff  agar  varied  with  the  composition 
of  the  glass  tubes  in  which  the  media  had  previously  been 
sterilized.  The  more  soluble  glasses  yielded  sufficient 
alkali  to  the  medium  to  inhibit  four-fifths  of  the  bacteria 
present  in  certain  cases. 

All  these  facts  make  it  evident  that  only  the  strictest 
adherence  to  a  standard  method  can  ensure  comparable 
results;  the  ordinary  nutrient  gelatin  should  then  in  all 
practical  sanitary  work  be  made  up  from  distilled 
water,  meat  infusion,  pepton  and  gelatin,  in  exact  ac- 


Quantitative  Bacteriological  Examination.         41 

cordance  with  the  directions  of  the  Standard  Methods 
Committee. 

Even  the  standard  procedure  fails  to  ensure  uniformity 
in  one  important  respect.  The  meat  infusion  which  it 
calls  for  is  in  itself  a  highly  variable  quantity.  Gage  and 
Adams  (1904),  in  the  examination  of  fifteen  lots  of  beef 
infusion,  found  variations  of  nearly  one  per  cent  in  organic 
solids  (calculated  on  the  weight  of  the  whole  infusion), 
after  the  final  filtration.  The  organic  constituents  of  the 
meat  infusion  varied,  therefore,  among  themselves  by 
nearly  the  total  amount  of  peptone  added.  It  is  to  be 
hoped  that  the  standard  methods  may  soon  be  so  revised 
as  to  eliminate  this  necessarily  uncertain  constituent  of 
nutrient  media.  Criticisms  of  detail  must,  however,  give 
way  to  the  importance  of  securing  fairly  comparable 
results;  and  the  confusion  which  would  follow  the  use  by 
individual  bacteriologists  of  media  made  without  meat 
would  outbalance  the  errors  inherent  in  the  standard 
procedure. 

Incubation.  —  Incubation  should  take  place  in  a  dark, 
well-ventilated  chamber  where  the  temperature  is  kept 
substantially  constant  at  20  degrees  and  where  the  atmos- 
phere is  practically  saturated  with  moisture.  It  has  been 
shown  by  Whipple  (Whipple,  1899)  and  others  that  the 
number  of  bacteria  developing  in  plate  cultures  is  to  an 
appreciable  extent  dependent  upon  the  presence  of  abun- 
dant oxygen  and  moisture.  Thus,  reckoning  the  number 
of  bacteria  developing  in  a  moist  chamber  at  100,  the 


42  Elements  of  Water  Bacteriology^ 

percentage  counts  obtained  in  an  ordinary  incubator  were 
as  follows:  75  when  the  relative  humidity  of  the  incubator 
was  60  per  cent  of  saturation;  82  when  it  was  75  per  cent; 
98  when  it  was  95  per  cent.  This  source  of  error  may  be 
avoided  by  the  use  of  ventilated  dishes  and  by  the  presence 
of  a  pan  of  water  in  the  incubating  chamber. 

According  to  American  and  German  practice,  plates 
made  for  sanitary  water  analysis  are  counted  at  the  end  of 
forty-eight  hours.  The  English  Committee  appointed  to 
consider  the  standardization  of  methods  for  the  Bac- 
terioscopic  Examination  of  Water  (1904)  fixed  the  time  at 
72  hours.  French  bacteriologists,  and  some  Germans 
(Hesse  and  Niedner,  1906),  still  recommend  longer  periods, 
and  the  table  on  p.  43  from  Miquel  and  Cambier  (Miquel 
and  Cambier,  1902)  shows  that  many  bacteria  fail  to 
appear  in  our  ordinary  procedure.  It  is,  however,  in  the 
main,  the  characteristic  water  bacteria  which  develop 
slowly,  sewage  bacteria  almost  without  exception  being 
rapid  growers.  The  longer  period  of  incubation  is, 
therefore,  not  only  inconvenient  but  undesirable,  since  it 
obscures  the  difference  between  good  and  bad  waters. 

Counting.  —  The  number  of  bacteria  is  determined  by 
counting  the  colonies  developed  upon  the  plate,  with  the 
aid  of  a  lens  magnifying  at  least  five  diameters.  For 
convenience  in  counting,  the  plate  may  be  placed  upon  a 
glass  plate  ruled  in  centimeter  squares  and  set  over  a  black 
tile,  or  the  tile  itself  may  be  ruled.  As  has  already  been 
said,  it  is  desirable  that  the  number  of  colonies  should  not 


Quantitative  Bacteriological  Examination.         43 

exceed  200,  for  when  the  number  is  very  high  the  colonies 
grow  only  to  a  small  size,  making  counting  laborious  and 
inaccurate,  and  many  do  not  develop  at  all.  The  best 
results  are  obtained  with  numbers  ranging  from  50 
to  200. 

EFFECT  OF  THE  LENGTH  OF  INCUBATION  OF  WATER 
BACTERIA  IN  GELATIN  UPON  THE  NUMBER  OF  COLO- 
NIES DEVELOPING. 

(MIQUEL  AND  CAMBIER,    1902.) 


Length  of  Incubation. 

Colonies 
Developed. 

Length  of  Incubation. 

Colonies 
Developed. 

i  day 

20 

9  days 

821 

2  days 

136 

10  days 

8  CQ 

3davs 

2^4 

ii  days  

UJV 
802 

•j*/0 

4daVS                          

387 

12  days  

O2I 

c  days 

^30 

13  days  . 

CKl 

6  days 

6?7 

14.  days 

O76 

7davs 

72  C 

\c  days  . 

tooo 

8  days 

780 

When  it  is  possible  to  do  so,  all  the  colonies  on  the  plate 
should  be  counted.  When  they  exceed  400  or  500  it  is 
often  easier,  and  fully  as  accurate,  to  count  a  fractional 
part  of  the  plate  and  estimate  the  total  number  therefrom. 
This  should  not  be  done,  however,  except  in  case  of 
necessity. 

It  is  customary  in  determining  numbers  to  make  plates 
in  duplicate,  thereby  affording  a  check  upon  one's  own 
work.  Owing  to  the  lack  of  precision  in  the  method,  the 
limit  of  experimental  error  is  a  wide  one.  It  should  be 


44 


Elements  of  Water  Bacteriology. 


possible  for  careful  manipulators  to  obtain  results  within 
10  per  cent  of  each  other,  but  a  closer  agreement  than  this 
is  hardly  to  be  expected.  It  has  been  suggested  by  the 
Committee  of  the  American  Public  Health  Association, 
that  the  following  mode  of  expressing  results  be  adopted 
in  order  to  avoid  the  appearance  of  a  degree  of  accuracy 
which  the  methods  do  not  warrant. 


501-1000 

1001-10,000 

10,001-50,000 

50,001-100,000 

100,001-500,000 

500,001-1,000,000 

1,000,001-5,000,000 


NUMBERS   OF  BACTERIA   FROM 

1-50  shall  be  recorded  to  the  nearest  unit 
51-100     «      "         «         "    "        "  Sl 

101-250     "      "         "         "     "        "  10 

251-500     "      "         "         "     "        "  25 

5° 

"       "  "  100 

"  "      "         500 

"  "        "  "  1,000 

"    "         '.'         10,000 
"        "        "    "        "        50,000 

"      "  "  100,000 


The  determination  of  numbers  of  bacteria  in  water  in 
the  field  has  frequently  been  attempted.  Since  the  labora- 
tory method  of  "plating  out"  is  difficult  to  use  in  field 
work,  the  Esmarch  tube  process  has  often  been  employed. 
This  consists  in  introducing  into  a  tube  of  melted  gelatin 
or  agar  i  c.c.  of  the  water,  and  then  rotating  the  tube  until 
the  medium  has  solidified  in  a  thin  layer  on  the  inner  wall. 
Other  bacteriologists  have  devised  ingenious  field  kits  for 
adapting  the  plate  method  to  this  purpose.  The  oppor- 
tunity for  air  infection  in  work  done  outside  a  proper 


Quantitative   Bacteriological  Examination.          45 

laboratory  is,  however,  always  great,  and  it  is  almost 
impossible  to  secure  proper  conditions  for  incubation  in 
any  temporary  establishment.  On  the  whole,  the  authors 
are  of  the  opinion  that  laboratory  examinations  are  to  be 
preferred  to  those  made  in  the  field,  if  a  laboratory  can  be 
reached  within  twelve  hours  of  the  time  of  collection  of 
the  samples. 


CHAPTER   III. 

THE    INTERPRETATION    OF    THE    QUANTITATIVE    BACTERI- 
OLOGICAL ANALYSIS. 

THE  information  furnished  by  quantitative  bacteri- 
ology as  to  the  antecedents  of  a  water  is  in  the  nature 
of  circumstantial  evidence  and  requires  judicial  interpre- 
tation. No  absolute  standards  of  purity  can  be  estab- 
lished which  shall  rigidly  separate  the  good  from  the  bad. 
In  this  respect  the  terms  "test"  and  "analysis"  so  univer- 
sally used  are  in  a  sense  inappropriate.  Some  scientific 
problems  are  so  simple  that  they  can  be  definitely  settled 
by  a  test.  The  tensile  strength  of  a  given  steel  bar,  for 
example,  is  a  property  which  can  be  absolutely  deter- 
mined. In  sanitary  water  examination,  however,  the 
factors  involved  are  so  complex,  and  the  evidence  neces- 
sarily so  indirect,  that  the  process  of  reasoning  much  more 
resembles  a  doctor's  diagnosis  than  an  engineering  test. 

The  older  experimenters  attempted  to  establish  arbi- 
trary standards,  by  which  the  sanitary  quality  of  a  water 
could  be  fixed  automatically  by  the  number  of  germs 
alone.  Thus  Miquel  (Miquel,  1891)  published  a  table 
according  to  which  water  with  less  than  10  bacteria  per 
c.c.  was  "excessively  pure,"  with  10  to  100  bacteria, 

46 


Quantitative  Bacteriological  Examination.        47 

"very  pure,"  with  100  to  1000  bacteria,  "pure,"  with 
1000  to  10,000  bacteria,  "mediocre,"  with  10,000  to 
100,000  bacteria, "  impure,"  and  with  over  100,000  bacteria, 
"very  impure."  Few  sanitarians  would  care  to  dispute  the 
appropriateness  of  the  titles  applied  to  waters  of  the  last 
two  classes;  but  many  bacteriologists  have  placed  the 
standard  of  "purity"  much  lower.  The  limits  set  by 
various  German  observers  range,  for  example,  from  50  to 
300.  Dr.  Sternberg  (Sternberg,  1892)  in  a  more  conserva- 
tive fashion,  has  stated  that  a  water  containing  less  than 
100  bacteria  is  presumably  from  a  deep  source  and  uncon- 
taminated  by  surface  drainage;  that  one  with  500  bacteria 
is  open  to  suspicion;  and  that  one  with  over  1000  bacteria  is 
presumably  contaminated  by  sewage  or  surface  drainage. 
This  is  probably  as  satisfactory  an  arbitrary  standard  as 
could  be  devised,  but  any  such  standard  must  be  applied 
with  great  caution.  The  source  of  the  sample  is  of  vital 
importance  in  the  interpretation  of  analyses;  a  bacterial 
count  which  would  condemn  a  spring  might  be  quite 
normal  for  a  river;  only  figures  in  excess  of  those  common 
to  unpolluted  waters  of  the  same  character  give  the  indi- 
cation of  danger.  Furthermore,  the  bacteriological  tests 
are  far  more  delicate  than  any  others  at  our  command, 
very  minute  additions  of  food  material  causing  an  immense 
multiplication  of  the  microscopic  flora.  This  delicacy 
necessarily  requires,  both  in  the  process  of  analysis  and 
the  interpretation  of  results,  a  Tiigh  degree  of  caution. 
As  pointed  out  in  the  previous  chapter,  the  touch  of  a 


48  Elements  of  Water  Bacteriology. 

• 

finger  or  the  entrance  of  a  particle  of  dust  may  wholly 
destroy  the  accuracy  of  an  examination.  Even  the 
slight  disturbance  of  conditions,  incident  upon  'the 
storage  of  a  sample  after  it  has  been  taken,  may  in  a  few 
hours  wholly  alter  the  relations  of  the  contained  microbic 
life.  It  is  necessary,  then,  in  the  first  place,  to  exercise 
the  greatest  care  in  allowing  for  possible  error  in  the 
collection  and  handling  of  bacteriological  samples;  and 
in  the  second  place,  only  well-marked  differences  in 
numbers  should  be  considered  significant. 

In  the  early  days  of  the  science,  discussion  ran  high  as 
to  the  interpretation  of  bacteriological  analysis,  and 
particularly  as  to  the  relation  of  bacterial  numbers  to 
the  organic  matter  present  in  a  water.  Different  observers 
obtained  inconsistent  results,  and  Bolton  (Bolton,  1886) 
concluded  that  there  was  no  relation  whatever  between 
the  chemical  composition  of  a  water  and  its  bacterial  con- 
tent. Tiemann  and  Gartner  (Tiemann  and  Gartner, 
1889)  furnished  the  key  to  the  difficulty  in  their  state- 
ment that  there  are  two  classes  of  bacteria,  the  great 
majority  of  species,  normally  occurring  in  the  earth 
or  in  decomposing  organic  matter,  which  require  abun- 
dance of  nutriment,  and  certain  peculiar  water  bacteria 
which  can  multiply  in  the  presence  of  such  minute 
traces  of  ammonia  as  are  present  in  ordinary  distilled 
water.  Even  these  prototrophic  or  semi-prototrophic 
forms  require  a  definite  amount  of  food  of  their  own 
kind. 


Quantitative  Bacteriological  Examination.       49 

Kohn  (1906)  determined  the  minimal  nutrient  material  re- 
quisite for  certain  of  the  latter,  and  found  that  they  could 
develop  in  the  presence  of  198  X  10  -  °  to  198  X  10  - 
per  cent  of  dextrose,  66  X  10  -  13  to  66  X  10  -  17  per  cent 
ammonium  sulphate  and  66  X  10  -  I3  to  66  X  10  -  L9  per 
cent  ammonium  phosphate.  Such  minute  amounts  of 
organic  matter  are  found  in  the  purest  of  natural  waters, 
and  under  exceptional  conditions  certain  species  of 
bacteria  may  therefore  multiply  in  bottled  samples,  or, 
at  times,  in  a  well  or  the  basin  of  a  spring.  In  normal 
surface-waters,  such  growths  of  the  prototrophic  forms 
do  not  apparently  occur.  Here  it  is  found  as  a  matter 
of  practical  experience  that  the  number  of  bacteria  present 
depends  upon  the  extent  to  which  the  water  has  been 
contaminated  with  decomposing  organic  matter,  either 
by  pollution  with  sewage  or  by  contact  with  the  surface 
of  the  ground.  The  bacterial  content  varies  as  the 
extent  and  character  of  the  contamination  varies.  It 
measures  not  merely  organic  matter  but  organic  matter 
in  a  state  of  active  decay,  and  like  the  ammonias  and 
other  features  of  the  sanitary  chemical  analysis,  indicates 
fresh  organic  pollution,  with  the  added  advantage  that 
the  presence  of  the  stable  nitrogenous  compounds  often 
present  in  peaty  waters  introduces  no  error  in  the  bac- 
teriological analysis. 

In  judging  of  a  surface-water  the  student  will  be  aided 
by  reference  to  the  figures  given  for  certain  normal  sources 
in  Chapter  I;  the  Boston  tap  water  with  50  to  200  bacteria 


5O  Elements  of  Water  Bacteriology. 

per  c.c.  (Philbrick,  1905)  and  the  water  of  Lake  Zurich 
with  an  average  of  71  in  summer  and  184  in  winter 
(Cramer,  1885)  may  be  taken  as  typical  of  good  potable 
waters;  and  numbers  much  higher  than  these  are  open 
to  suspicion,  since  all  contamination  whether  contributed 
by  sewage  or  by  washings  from  the  surface  of  the  ground 
is  a  possible  source  of  danger.  The  excess  of  bacteria 
in  surface-waters  during  the  spring  and  winter  months  is 
by  no  means  an  exception  to  the  general  rule  that  high 
numbers  are  significant,  since  the  peril  from  supplies  of 
this  character  is  clearly  shown  by  the  spring  epidemics  of 
typhoid  fever  which  at  the  times  of  melting  snow  visit 
communities  making  use  of  unprotected  surface-waters. 
Streams  receiving  direct  contributions  of  sewage  exhibit  a 
similar  excess  of  bacteria  at  all  times,  numbers  rising  to  an 
extraordinary  height  near  the  point  of  pollution  and  fall- 
ing off  below  as  the  stream  suffers  dilution  and  the  sewage 
organisms  perish.  Miquel  (Miquel,  1886)  records  300 
bacteria  per  c.c.  in  the  water  of  the  Seine  at  Choisy,  above 
Paris;  1 200  at  Bercy  in  the  vicinity  of  the  city,  and  200,000 
at  St.  Denis  after  the  entrance  of  the  drainage  of  Paris. 
Prausnitz  (Prausnitz,  1890)  found  531  bacteria  per  c.c. 
in  the  Isar  above  Munich,  227,369  near  the  entrance  of 
the  principal  sewer,  9111  at  a  place  13  kilometers  below 
the  city,  and  2378  at  Freising,  20  kilometers  further 
down.  Jordan  (Jordan,  1900),  in  his  study  of  the  fate  of 
the  sewage  of  Chicago,  found  1,245,000  bacteria  per  c.c. 
in  the  drainage  canal  at  Bridgeport,  650,000  twenty-nine 


Quantitative  Bacteriological  Examination.  51 

miles  below  at  Lockport,  and  numbers  steadily  decreasing 
below  to  3660  at  Averyville,  159  miles  below  the  point  of 
original  pollution.  Below  Averyville  the  sewage  of  Peoria 
enters  and  the  numbers  rise  to  758,000  at  Wesley  City, 
decreasing  to  4800  in  123  miles  flow  to  Kampsville. 
Brezina  (1906)  found  1900  bacteria  per  c.c.  in  the  Donau 
River  above,  and  110,000  at  the  mouth  of,  the  Donau 
canal.  This  number  fell  to  85,000  one  kilometer  below, 
62,000  four  kilometers  below,  and  40,000  seven  kilometers 
down  the  stream.  Vincent  (1905)  records  from  1000  to 
46,000  bacteria  per  c.c.  in  the  waters  of  more  or  less 
polluted  French  rivers.  Mayer  (1902),  on  the  other 
side  of  the  world,  found  21  and  35  bacteria  per  c.c.  in  the 
Shaho  River,  near  its  source,  in  the  vicinity  of  the  great 
Chinese  Wall,  and  from  100,000  to  600,000  in  the  highly 
polluted  Whangpo,  near  its  mouth. 

In  ground-waters  we  have  seen  that  bacteria  may  occa- 
sionally be  present  in  considerable  numbers,  but,  if  so,  they 
are  generally  organisms  of  a  peculiar  character,  incapable 
of  development  on  the  ordinary  nutrient  media  in  the 
standard  time.  Thus  in  forty-eight  hours  we  often  obtain 
counts  measured  only  in  units  or  tens  such  as  have  been 
recorded  in  Chapter  I.  When  higher  numbers  are 
present,  the  general  character  of  the  colonies  must  be 
taken  into  account,  since  besides  the  slowly-growing 
forms  certain  other  water  bacteria,  which  require  a  com- 
paratively small  amount  of  nutriment,  may  multiply  at 
times  in  a  deep  well  or  the  basin  of  a  spring.  In  such  a 


52  Elements  of  Water  Bacteriology. 

case,  however,  the  appearance  of  the  plates  at  once  reveal, 
the  peculiar  conditions,  for  the  colonies  are  of  one  kind 
and  that  distinct  from  any  of  the  sewage  species.  Thus 
Dunham  (Dunham,  1889)  reports  that  the  mixed  water 
from  a  series  of  driven  wells  gave  2  bacteria  per  c.c., 
while  another  well,  situated  just  like  tjie  others,  contained 
5000,  all  belonging  to  a  single  species  common  in  the  air. 
Except  in  such  peculiar  cases  as  this,  high  numbers  in  a 
ground-water  mean  contamination. 

The  process  of  slow  sand  nitration  for  the  purification 
of  unprotected  surface-waters  is  essentially  similar  to  the 
action  which  takes  place  in  nature  when  rain  soaks  through 
the  ground  to  appear  in  wells  and  springs;  and  it  is  in  the 
examination  of  the  effluent  from  such  municipal  plants 
that  the  quantitative  bacteriological  analysis  finds,  per- 
haps, its  most  important  application.  The  chemical 
changes  which  occur  in  the  passage  of  water  through 
sand  at  a  rate  of  1,000,000  or  2,000,000  gallons  per  acre 
per  day  are  so  slight  as  to  be  negligible.  The  bacteria 
present  should,  however,  suffer  a  reduction  of  98  or  99  per 
cent,  and  their  numbers  furnish  the  best  standard  for 
measuring  the  efficiency  of  such  filtration  plants.  At 
Lawrence,  in  1905,  Clark  found  an  average  of  12,700  bac- 
teria per  c.c.  in  the  raw  water  of  the  Merrimac  River,  while 
the  number  present  in  the  filtered  water  was  only  70 
(Massachusetts  State  Board  of  Health,  1906).  Where 
the  number  of  bacteria  in  the  applied  water  is  smaller  it 
is  difficult  to  obtain  so  high  a  percentage  efficiency.  At 


Quantitative  Bacteriological  Examination.  53 

Washington,  for  example,  prolonged  sedimentation  gener- 
ally reduces  the  bacterial  numbers  to  less  than  a  thou- 
sand, and  it  is  almost  impossible  to  secure  a  99  per  cent 
removal.  The  actual  numbers  of  bacteria  in  the  effluent 
are,  however,  much  lower  than  at  Lawrence.  The 
monthly  average  results  obtained  for  a  year  at  these  two 
plants  are  tabulated  on  page  54. 

Mechanical  nitration  gives  similar  results.  Fuller  at 
Cincinnati  (Fuller,  1899)  records  27,200  organisms  per  c.c. 
in  the  water  of  the  Ohio  River  between  September  21, 
1898,  and  January  25,  1899,  while  the  average  content  of 
the  effluent  from  the  Jewell  filter  was  400.  Data  with 
regard  to  the  operation  of  mechanical  filters  are  now 
abundant,  since  all  over  the  world  the  operation  of  these 
plants  is  controlled  by  bacteriological  methods.  Recently 
Johnson  (1907)  has  reported  some  interesting  results  from 
the  far  East.  At  Osaka,  Japan,  an  average  of  200  bac- 
teria per  c.c.  in  the  raw  water  of  the  Yodo  River  was 
reduced,  in  1905,  to  an  average  of  25  by  slow  sand 
filters;  at  Bethmangala,  India,  in  1906,  mechanical  filters 
treated  the  water  of  the  Palar  River,  containing  4350  bac- 
teria per  c.c.,  and  yielded  an  effluent  with  only  13  per  c.c. 
(Johnson,  1907). 

The  average  monthly  results  obtained  with  the  new 
mechanical  filter  plant  at  Harrisburg,  Pa.,  are  included  in 
the  table  on  page  54  for  comparison  with  the  figures 
recorded  at  Washington  and  Lawrence;  and  these  may  be 
taken  as  typical  since  the  Harrisburg  plant  is  the  latest 


54 


Elements  of  Water  Bacteriology. 


of  its  type,  as  the  Washington  plant  is  the  newest  and 
most  perfectly  equipped  of  slow  sand  filters. 

REMOVAL  OF  BACTERIA  BY  NATURAL  SAND  FILTERS 
AND  MECHANICAL  FILTERS.  BACTERIA  PER  C.C.  IN 
APPLIED  WATER  AND  EFFLUENT. 

MONTHLY  AVERAGES. 


Washington,  1906. 

Lawrence,  1905. 

Harrisburg,   1906. 

Applied 
Water. 

Effluent. 

Applied 
Water. 

Effluent. 

Applied 
Water. 

Effluent. 

Jan.      . 

1500 

39 

14,200 

no 

951° 

104 

Feb.      . 

55° 

16 

14,800 

55 

21,228 

298 

Mar.     . 

650 

19 

10,300 

55 

3i>326 

75 

Apr. 

400 

22 

3600 

170 

39.905 

42 

May 

65 

17 

1900 

12 

6187 

86 

June     . 
July      . 

220 
1  60 

17 
26 

9600 
3900 

9 

55 

2903 
685 

3i 

10 

Aug.     . 

IQO 

14 

19,500 

37 

1637 

5 

Sept.     . 

130 

14 

i3>5°° 

44 

836 

12 

Oct.      . 

275 

16 

39,800 

no 

7575 

63 

Nov.     . 

220 

12 

8,700 

70 

26,224 

236 

Dec.     . 

700 

45 

37>525 

I63 

In  well-managed  purification  plants  the  bacteria  in  the 
effluent  are  determined  daily,  and  any  deviation  from  the 
normal  value  at  once  reveals  disturbing  factors  which  may 
impair  the  efficiency  of  the  process.  In  Prussia  official 
regulations  demand  such  systematic  examinations,  and 
prescribe  50  as  the  maximum  number  of  bacteria  allowable 
in  the  filtered  water.  In  the  same  way  the  condition  of 
an  unpurified  surface  supply  may  be  determined  by  daily 
bacteriological  analyses  and  warnings  of  danger  issued 
to  the  public,  as  has  been  done  at  Chicago  and  other 


Quantitative  Bacteriological  Examination.         55 

cities.  In  general,  any  regular  determination  of  variations 
from  a  normal  standard  furnishes  ideal  conditions  for  the 
bacteriological  methods;  and  the  detection  by  Shuttle  worth 
(Shuttleworth,  1895)  of  a  break  in  a  conduit  under  Lake 
Ontario  by  a  rise  in  the  bacteria  of  the  Toronto  water- 
supply  may  be  cited  as  a  classic  example  of  its  application. 

Often,  however,  the  expert  is  called  to  pass  upon  the 
character  of  a  water  of  which  no  series  of  analyses  is  avail- 
able and  whose  surroundings  it  may  be  impossible  for  him 
to  inspect.  It  has  been  said  that  single  bacteriological 
analyses  of  this  kind  are  valueless;  but  this  we  believe 
cannot  always  be  maintained.  Knowing  the  normal 
bacterial  range  for  a  given  class  of  waters,  even  an  isolated 
analysis  may  show  such  an  excess  as  to  have  great  sig- 
nificance, as  a  few  practical  examples  will  make  clear 
(Winslow,  1901). 

In  the  spring  of  1900  the  city  of  Hartford,  Conn.,  was 
using  a  double  supply,  from  the  Connecticut  River  and 
from  a  series  of  impounding  reservoirs  among  the  hills. 
A  single  series  of  plates  showed  from  4000  to  7000  bac- 
teria per  c.c.  in  the  water  of  the  river,  while  the  reservoir 
water  contained  300  to  900.  The  abandonment  of  the 
river  supply  followed,  and  at  once  the  excessive  amount 
of  typhoid  fever  in  the  city  was  curtailed. 

In  the  fall  of  1900,  Newport,  R.  L,  experienced  an  out- 
break of  typhoid  fever,  and  when  suspicion  was  thrown 
upon  the  surface  water-supply,  chemical  analysis  of  the 
latter  was  not  wholly  reassuring;  but  there  were  only  334 


56  Elements  of  Water  Bacteriology. 

bacteria  per  c.c.  in  the  water  from  the  taps,  while  a  well 
in  the  infected  district  gave  6100.  It  was  no  surprise 
to  find,  on  a  further  study  of  the  epidemic,  that  the  well 
was  largely  at  fault  and  the  public  supply  was  not. 

In  the  case  of  ground-water  the  evidence  is  usually  even 
more  distinct.  At  Framingham,  Mass.,  in  1903,  high 
chlorin  content  in  the  public  supply,  drawn  from  a  filter 
gallery  beside  a  lake,  had  led  to  public  anxiety.  Five 
samples  from  different  parts  of  the  system  showed  averages 
of  i,  2,  2,  2,  and  4  bacteria  per  c.c.;  and  taking  this  in 
conjunction  with  the  other  features  of  the  bacteriological 
analysis,  it  was  possible  to  report  that  any  pollution 
introduced  upon  the  gathering  ground  had  at  the  time  of 
examination  been  entirely  removed. 


CHAPTER    IV. 

DETERMINATION     OF     THE     NUMBER     OF     ORGANISMS 
DEVELOPING  AT  THE  BODY  TEMPERATURE. 

THE  count  of  colonies  upon  the  gelatin  plate  measures, 
as  we  have  pointed  out,  the  number  of  the  metatrophic 
bacteria  in  general;  and  the  distribution  of  these  forms 
corresponds  with  the  decomposition  of  organic  matter 
wherever  it  may  occur.  In  this  great  class  there  are 
some  species  which  will  grow  under  a  wide  variety  of 
conditions.  These  are  present  in  most  waters  in  small 
numbers,  and  in  sources  containing  much  decaying  vege- 
table matter  they  occur  in  abundance.  Other  meta- 
trophic forms,  however,  through  a  semi-parasitic  mode 
of  life  have  become  specially  adapted  to  the  peculiar 
conditions  characteristic  of  the  animal  body;  and  these 
bacteria  possess  the  property  of  developing  most  actively 
at  the  temperature  of  the  human  organism,  37°  C.,  which 
altogether  checks  the  growth  of  the  majority  of  normal 
earth  and  water  forms.  The  determination  of  the  num- 
ber of  organisms  growing  at  the  body  temperature  may 
throw  light,  then,  on  the  presence  of  direct  sewage  pollu- 
tion, since  the  bacteria  from  the  alimentary  canal  flourish 

57 


58  Elements  of  Water  Bacteriology. 

under  such  conditions,  while  most  of  those  derived  from 
other  sources  do  not.  Savage  classifies  the  bactena 
which  may  be  found  in  water  under  three  headings": 
normal  inhabitants,  like  B.  fluorescens;  unobjectionable 
aliens  (from  soil),  like  B.  mycoides,  and  objectionable 
aliens  (from  excreta),  like  B.  coli.  The  first  sort  and 
many  of  the  second  sort  are  generally  unable  to  grow  at 
37  degrees.  This  criterion  is  not  an  absolute  one.  Savage 
(1906)  reports  an  experiment  in  which  unpolluted  soil, 
which  had  not  been  manured  or  cultivated  for  at  least 
three  years,  was  added  to  tap  water,  with  the  result  that  a 
20°  count  of  76  was  increased  to  1970,  and  a  37°  count  of  3 
was  raised  to  1630.  In  this  case  most  of  the  bacteria  in 
the  soil  were  capable  of  development  at  body  tempera- 
ture. Experience  shows,  however,  that  the  numbers  of 
such  bacteria  which  actually  reach  natural  waters  from 
such  sources  are  seldom  large.  The  count  at  37  degrees, 
therefore,  helps  to  distinguish  contamination  by  wash  of 
the  soil  of  a  virgin  woodland  from  pollution  by  excreta, 
since  in  the  former  case  the  proportion  of  blood-tempera- 
ture organisms  is  much  smaller  than  in  the  latter.  Further- 
more, this  method  is  free  from  much  of  the  error  introduced 
by  the  multiplication  of  bacteria  after  the  collection  of  a 
sample,  as  most  of  the  forms  which  grow  in  water  during 
storage  cannot  endure  the  higher  temperature  and  conse- 
quently do  not  develop  upon  incubation.  Recently,  for 
example,  water  from  a  spring  of  good  quality  was  shipped 
to  the  laboratory  from  a  considerable  distance.  Gelatin 


Body   Temperature  Organisms.  59 

plates  showed  4200  bacteria  per  c.c.,  but  agar  plates  at  37 
degrees  were  sterile. 

The  body-temperature  count  must,  of  course,  be  made 
upon  agar  plates,  otherwise  the  procedure  is  the  same 
which  has  already  been  described  for  the  routine  quan- 
titative bacteriological  analysis.  The  period  of  incuba- 
tion ordinarily  adopted  by  the  writers  is  twenty-four 
hours,  as  little  development  occurs  after  that  time.  Diffi- 
culty is  sometimes  caused  by  the  spreading  of  colonies 
of  certain  organisms  over  the  surface  of  the  plate  in  the 
water  of  condensation  which  gathers;  this  may  be  avoided 
by  inverting  the  plates  after  the  agar  is  once  well  set  or 
still  better  by  the  use  of  plates  provided  with  earthenware 
tops  as  suggested  by  Hill.  The  porous  earthenware 
absorbs  the  water  which  condenses  on  it,  the  surface  of 
the  plate  remains  comparatively  dry,  and  the  percentage 
of  "  spread  "  plates  is  reduced  from  30  per  cent  to  i  per 
cent  (Hill,  1904). 

Additional  evidence  as  to  the  character  of  a  water 
sample  may  be  obtained  with  little  extra  trouble  by  add- 
ing a  sugar  and  some  sterile  litmus  to  the  agar  medium 
and  observing  the  fermenting  powers  of  the  organisms 
present,  as  first  suggested  by  Wurtz  (Wurtz,  1892)  for  the 
separation  of  B.  coli  from  B.  typhi.  It  happens  that  the 
most  abundant  intestinal  organisms,  belonging  to  the 
groups  of  the  colon  bacilli  and  the  streptococci,  decom- 
pose dextrose  and  lactose  with  the  formation  of  a  large 
excess  of  acid.  The  decomposition  of  the  latter  sugar 


60  Elements  of  Water  Bacteriology. 

on  the  other  hand  is  almost  entirely  wanting  among  the 
commoner  saprophytic  bacteria,  and  therefore  lactose  is 
most  commonly  used  in  making  sugar  agar,  i  per  cent 
being  added  to  the  medium  just  before  the  final  filtration 
(between  steps  15  and  16  in  the  standard  process  of  media 
making  given  on  p.  209).  In  pouring  the  plate  a  cubic 
centimeter  of  sterile  litmus  solution  should  be  added. 
After  incubation  the  colonies  of  the  acid-forming  organ- 
isms will  be  clearly  picked  out  by  the  reddening  of  the 
adjacent  agar.  Only  those  colonies  which  are  sharply 
colored  should  be  considered  as  significant,  since  certain 
bacteria  of  the  hay-bacillus  group  produce  weak  acid 
and  faint  coloring  of  the  litmus. 

When  polluted  waters  are  examined  in  this  manner  the 
number  of  organisms  developing  on  the  lactose-agar  plate 
will  be  very  high,  almost  equalling  in  some  cases  the  total 
count  obtained  on  gelatin.  Chick  (Chick,  1901),  using 
a  lactose-agar  medium  with  the  addition  of  one-thousandth 
part  of  phenol,  found,  of  colon  bacilli  alone,  6100  per  c.c. 
in  the  Manchester  ship  canal,  55  to  190  in  the  polluted 
River  Severn,  and  numbers  up  to  65,000  per  gram  in 
roadside  mud.  In  an  examination  of  water  from  the 
Charles  River  above  Boston,  total  37-degree  counts 
ranging  from  9800  to  16,900  have  been  found.  The 
average  result  of  56  examinations  of  Boston  sewage  from 
July  to  December,  1903,  showed  5,430,000  bacteria  per 
c.c.  at  20  degrees,  and  3,760,000  per  c.c.  at  37  degrees, 
of  which  1,670,000  were  acid  formers.  The  average  of 


Body  Temperature  Organisms. 


61 


25  samples  examined  in  July  and  August,  1904,  showed 
1.690,000  bacteria  per  c.c.  at  20  degrees  and  1,400,000  at 
37  degrees,  while  429,000  per  c.c.  were  acid  formers 
(Winslow,  1905). 

In  unpolluted  waters  not  only  the  absolute  number  of 
organisms  developing  at  the  body  temperature,  but  the 
ratio  to  the  gelatin  count,  is  very  different.  Rideal  (Rideal, 
1902)  states  that  the  proportion  between  the  two  counts 
in  the  case  of  a  London  water  in  a  year's  examination 
was  on  the  average  one  to  twelve.  Mathews  (Mathews, 
1893)  in  1893,  gave  the  following  figures,  the  contrast 
between  the  ponds  and  streams,  which  were  presumably 
exposed  to  pollution,  on  the  one  hand,  and  the  wells, 
springs,  and  taps,  on  the  other,  being  marked. 


Source  of  Water. 

Average  I 
Colonies 

[umber  of 
per  c.c. 

Gelatin,  20°. 

Lactose-  Agar, 
37.5°. 

^Vells   springs             ...        

1664 

28 

Reservoirs       

IC7 

AT. 

Ponds 

2Q6 

Q<\ 

Taps 

242 

24 

Streams 

273 

IOI 

According  to  the  English  Committee  appointed  to 
consider  the  Standardization  of  Methods  for  the  Bacterio- 
scopic  Examination  of  Water  (1904),  the  ratio  of  the  20- 
degree  count  to  the  3  7 -degree  count  in  good  waters  is 


62 


Elements  of  Water  Bacteriology. 


RELATION  OF  20°  AND  37°  COUNTS  IN  SAMPLES  OF  WATER 

FROM   APPARENTLY   UNPOLLUTED   SOURCES. 

(WINSLOW  AND  NI BECKER,  1903  ) 


Source  of  Samples. 

Number  of  Samples.  | 

Gela- 
in 
Plates. 

20°. 

li 

$2 

r 

Litmus- 
lactose- 
agar 
Plates, 

37°. 

Dextrose  Broth 
Tubes. 

Average  Number 
of  Colonies. 

Plates  Showing 
Red  Colonies. 

Number  of  Tubes. 

*, 

•8<S 

I* 

§ 

& 

Jf 

iJ 

is 
ig* 

8  M 

!i 

1' 

Cambridge  supply  (tap)     .    . 
Wakefield  and  Stoneham  sup- 
ply (tap)    . 

5 

6 
i 
6 

3 
6 

6 
5 

4 
3 
5 

2 

I 

61 
15 

32 
i5 

i 

22 
22 
10 
I 

2 

6 

i 
3 

7 

i 

94 

59 
16 

35 
141 
3,717 
36 
232 

13 

738 
524 
4,700 

223 
18 
294 
167 

'365 
181 
811 

47 
188 

i,235 
269 

i5 

II 
6 
18 

2 
21 

I 

9 
14 

2 
46 

8 
o 

12 

7 

i 

2 

9 

4 
3i 
3 
4 
o 

0 
2 

27 

6 

2 

I 

o 

0 
0 
2 
0 
0 

o 

0 
0 
0 
2 
0 
0 
0 
0 
0 
0 
0 

o 

0 
0 
0 

o 

0 

o 

0 
0 

o 

0 

15 

21 

18 

3 
18 

9 
18 
18 
15 

12 

9 
15 
6 

4 

45 
95 

45 

636 
65 

3° 

i 

18 

3 
9 

21 

3 

0 
0 

o 

0 
0 
2 
0 
0 
0 
0 

3 
o 

0 
0 

13 

o 

13 

I 

0 
2 
0 
2 
O 
0 
0 

o 
5 

0 
0 

0 
0 

o 

0 
0 
2 
0 
0 
0 
0 
0 
0 
0 
0 

13 

0 

13 

I 
o 

2 
0 
2 

o 

0 
0 

o 
5 

0 

0 

o 
o 

0 

o 

0 

o 

0 

o 

0 
0 

3 
o 
o 

0 
0 

o 

0 

o 

0 
0 
0 
0 
0 
0 
0 

0 
0 

0 

D 

3 

Lynn  supply  (tap)  
Brookline  supply  (tap)  .  .  . 
Plymouth  supply  (tap)  .  .  . 
Peabody  supply  (tap)  .... 
Dedham  supply  (tap).  .  .  . 
Newburyport  supply  (tap)  . 
Salem  supply  (tap)  .... 
Taunton  supply  (tap)  .  .  . 
Sharon,  (well)  (tap) 

Medford  supply  (tap)     .    .    . 
Milton  supply  (tap)    .... 
Westerly,  R.  I.,  supply  (tap) 
Brooks  
Driven  wells  

Springs  

Ponds  fed  by  brooks  .... 
M^elted  snow  .  . 

Pools  in  fields 

Pools  in  woods  

Roadside  pools     
Stream,  Blue  Hill  Reservation 
Flow  from  rocks      
Ponds  fed  by  springs      .    .    . 
Drainage  from  manured   pas- 
ture 

Swamps 

Rain-water  after  twelve  hours' 
heavy  fall      
Shallow  well  in  Lynn  woods. 
Totals    

259 

4 

775 

41 

38 

Body  Temperature  Organisms.  63 


generally  considerably  higher  than  10  to  i.  "With  a 
polluted  water  this  ratio  is  approached,  and  frequently 
becomes  10  to  2,  10  to  3  or  even  less." 

In  1903  Nibecker  and  one  of  ourselves  (Winslow  and 
Nibecker,  1903)  made  an  examination  of  259  samples 
of  water  from  presumably  unpolluted  sources  in  Eastern 
Massachusetts,  including  public  supplies,  brooks,  springs, 
ponds,  driven  wells,  and  pools  in  the  fields  and  woods, 
with  a  view  to  testing  the  value  of  the  body-temperature 
examination.  In  many  cases  the  samples  showed  high 
gelatin  counts,  since  some  of  the  waters  were  exposed  to 
surface  wash-  from  vacant  land,  but  the  average  number 
of  organisms  developing  on  lactose  agar  at  37  degrees  was 
less  than  8  per  c.c.  The  highest  individual  counts  obtained 
were  95  in  a  meadow  pool,  83  in  a  brook,  and  74  in 
a  barnyard  well,  the  latter  probably  actually  polluted. 
Only  two  samples  in  the  whole  series,  one  from  the  well 
above  mentioned,  gave  any  red  colonies  on  the  agar 
plates. 

Important  data  as  to  the  distribution  of  bacteria  which 
will  develop  at  high  temperatures  may  be  found  in  a  recent 
paper  by  Gage  (1906),  coupled  with  a  suggestive  discussion 
of  the  general  significance  of  bacterial  ratios.  The  table 
on  p.  64  shows  some  of  the  most  significant  results  obtained 
by  plating  waters  of  various  degrees  of  purity  at  20,  40  and 
50  degrees.  We  have  rearranged  the  lines  of  the  table  so 
as  to  make  the  progression  from  more  to  less  polluted  waters 
a  fairly  regular  one.  The  colony  count  at  50  degrees 


64 


Elements  of  Water  B.acteriology. 


AVERAGE  NUMBER  OF  BACTERIA  AND  ACID-PRODUCERS 
DEVELOPING  AT  20°,  40°  AND  50°  C.  WITH  DIFFERENT 
CLASSES  OF  WATERS. 

(GAGE,  1906.    REARRANGED.) 


Bacteria  per  c.c. 

Acid-producing  Bacteria. 

20°  C. 

4D. 

40°  C. 
24  Hr. 

50°  C. 
24  Hr. 

20°  C. 

4D. 

40°  C. 
24  Hr. 

50°  C. 
24  Hr. 

Sewage    .... 

2,990,000 

557.500 

7700 

1,940,000 

346,000 

4400 

it 

1,676,000 

360,000 

29,500 

1,032,000 

283,000 

24,900 

Septic  effluent    . 
Contact  effluent 

485,000 
146,600 

126,500 
26,100 

410 

8300 

241,000 
112,400 

90,000 
22,700 

240 

8000 

«             « 

389,000 

59»3°o 

8000 

292,000 

45,000 

8000 

«             « 

306,000 

89,600 

485 

193,000 

46,000 

200 

Trickling  filter 

effluent    .    .    . 

I5.SOO 

1730 

154 

15,200 

1360 

100 

«            « 

23,300 

2030 

54 

16,000 

1180 

20 

Canal  water    .    . 

16,400 

112 

5 

6700 

87 

2 

River  water    .    . 

16,900 

207 

4 

2500 

134 

2 

Settled        canal 

water    .... 

2800 

212 

2 

1650 

66 

I 

Sand   filter  efflu- 

ent (sewage)   . 

1640 

1375 

2 

2360 

H95 

I 

«       «       « 

35 

4 

0 

29 

2 

O 

«       «       <( 

1300 

I30 

I 

345 

119 

O 

«       n      n 

670 

170 

2 

1045 

J54 

o 

Water  filter  efflu- 

ent   .        ... 

32 

^ 

I 

6 

i 

I 

«       «      « 

o 
7*5 

170 

2 

259 

101 

I 

«       «       n 

62 

I 

0 

16 

o 

0 

n      «       ii 

150 

22 

I 

14 

17 

I 

it      «       n 

64 

5 

I 

ii 

3 

I 

Shallow  well   .    . 

IOOO 

2 

0 

3 

i 

o 

«          « 

507 

72 

0 

82 

55 

o 

Pond 

27 

I 

o 

8 

i 

0 

/ 

71 

8 

o 

3° 

5 

0 

Spring      .... 

49 

0 

o 

6 

o 

0 

«« 

80 

2 

o 

8 

2 

o 

Driven  well     .    . 

4i 

0 

0 

o 

O 

o 

Body  Temperature  Organisms.  65 

shows  an  even  sharper  differentiation  than  that  at  40 
degrees.  Gage  rightly  concludes  that  "the  information 
to  be  obtained  by  counts  of  bacteria  and  acid-producing 
organisms  at  any  one  of  the  above  temperatures  is  greatly 
increased  by  the  combination  of  the  results  obtained  from 
counts  at  two  or  more  temperatures. " 

In  warm  weather  the  interpretation  of  the  body  tempera- 
ture count  must  be  made  less  rigid  than  at  other  seasons. 
Recent  investigations  have  shown  that  in  midsummer 
bacteria  capable  of  growth  at  37  degrees  are  more  abun- 
dant in  normal  waters  than  in  winter  and  spring. 

Winslow  and  Phelps  (in  a  recent  unpublished  study) 
examined  86  samples  from  springs,  wells,  brooks  and 
pools  during  the  winter  and  spring  months  and  found  only 
12  which  showed  more  than  25  bacteria  per  c.c.  and  only 
3  which  showed  mo're  than  100  per  c.c.  on  lactose-agar. 
On  the  other  hand,  of  58  samples  from  ,  corresponding 
sources  examined  in  summer,  16  contained  more  than  100 
bacteria  per  c.c.  A  series  of  20  pools,  ponds  and  brooks 
at  Mt.  Desert,  Maine,  which  were  entirely  free  from 
human  or  animal  pollution,  were  examined  in  the  late 
summer  of  1906.  Only  four  of  the  20  samples  gave 
counts  under  25  at  37  degrees,  and  seven  of  them  gave 
counts  over  100,  the  highest  figure  being  425.  Similarly 
it  has  been  pointed  out  that  in  tropical  countries  organisms 
capable  of  development  at  37  degrees  may  thrive  abun- 
dantly in  normal  waters. 

A  majority  of  the  English   Committee  appointed    to 


66  Elements  of  Water  Bacteriology. 

consider  the  standardization  of  methods  for  the  Bao 
terioscopic  Examination  of  Water  (1904),  recommended 
the  body  temperature  count  as  a  standard  procedure; 
the  American  Committee  on  Standard  Methods  of  Water 
Analysis  (1905)  failed  to  adopt  this  method  in  its  last 
report.  It  is  to  be  hoped  that  it  will  see  fit  to  do  so  in 
the  future;  and  meanwhile  individual  bacteriologists  will 
find  it  of  much  service  in  supplementing  the  20-degree 
determination  on  gelatin.  Under  ordinary  conditions  it 
is  clear  that  organisms  growing  at  the  body  temperature 
and  those  fermenting  lactose  are  not  numerous  in  normal 
waters.  The  absolute  count  at  37  degrees  seldom  exceeds 
50,  and  is  rarely  over  10  per  cent  of  the  20-degree  count, 
except  after  hot  periods  in  the  late  summer;  acid  pro- 
ducers are  generally  entirely  absent.  On  the  other  hand, 
the  numbers  on  the  litmus-lactose-agar  plate  will  be 
likely  to  run  into  hundreds  with  a  good  proportion  of  red 
colonies  when  polluted  waters  are  examined. 


CHAPTER  V. 

THE  ISOLATION  OF  SPECIFIC  PATHOGENES  FROM 
WATER. 

THE  discovery  of  the  organisms  which  specifically  cause 
infectious  diseases  naturally  led  to  the  hope  that  their 
isolation  from  polluted  water  might  become  the  most 
convincing  proof  of  its  sanitary  quality.  The  typhoid 
bacillus  and  the  spirillum  of  Asiatic  cholera  were  in  this 
connection  of  paramount  importance,  and  to  the  search 
for  them  many  investigators  devoted  themselves. 

In  the  earlier  examinations  of  water  for  the  typhoid 
bacillus  an  attempt  was  made  either  to  use  media,  which 
especially  favored  the  growth  of  the  microbe  sought  for, 
or  to  begin  with  some  process  of  "enrichment"  in  which 
the  sample  was  incubated  under  conditions  which  would 
favor  the  -growth  of  the  pathogenic  organisms  while  check- 
ing the  development  of  the  common  water  bacteria.  It 
was  apparent  that  the  body  temperature  and  the  presence 
of  a  slight  excess  of  free  acid  furnished  such  conditions, 
and  most  of  the  methods  suggested  rest  upon  these  prin- 
ciples. Among  these,  one  of  the  earliest  was  that  of  Parietti 
(Parietti,  1890),  which  consists  in  the  addition  of  portions 
of  the  water  to  a  series  of  broth  tubes  containing  increasing 

67 


68  Elements  of  Water  Bacteriology. 

amounts  of  a  solution  of  4  per  cent  hydrochloric  acid  and 
5  per  cent  phenol.  From  tubes  in  which  growth  occurs 
after  twenty-four  hours  at  37  degrees,  the  organisms 
present  may  be  isolated  in  pure  cultures  by  some  plating 
method  and  identified  by  subcultures. 

The  great  difficulty  with  a  majority  of  the  enrichment 
processes  is  that  the  conditions  which  favor  the  multipli- 
cation of  the  typhoid  bacillus  are  frequently  suited  in  an 
even  higher  degree  to  B.  coli  and  other  intestinal  organ- 
isms. Being  present  in  almost  all  cases  in  much  higher 
numbers  than  B.  typhi,  these  germs  develop  abundantly, 
and  effectually  mask  any  disease  germs  originally 
present.  In  order  to  obviate  this  difficulty,  Hankin 
(Hankin,  1899),  after  adding  successively  increasing 
portions  of  Parietti  solution  to  tubes  inoculated  with  the 
water  to  be  tested,  selected  the  second  highest  tube  of  the 
series  in  which  growth  occurred  for  the  inoculation  of  a  new 
set,  finally  plating  as  above.  He  believed  that  the  chance 
for  overgrowth  in  this  method  was  somewhat  decreased, 
but  in  the  hands  of  other  investigators  it  has  not  met 
with  marked  success.  Klein  (Thomson,  1894)  in  his 
investigations,  made  use  of  the  Berkefeld  filter  to  con- 
centrate the  organisms  in  the  sample.  Some  observers 
have  abandoned  the  enrichment  process  altogether  and 
recommend  direct  plating  upon  phenolated  gelatin  or 
on  the  Eisner  (Eisner,  1896)  medium  made  by  adding  10 
per  cent  of  gelatin  and  i  per  cent  of  potassium  iodide  to 
an  infusion  of  potato  whose  reaction  has  been  adjusted 


Isolation  of  Specific  Pathogenes.  69 

to  30  on  Fuller's  scale.  In  all  cases,  however,  the  chance 
of  success  is  small,  as  is  well  shown  by  the  experiments 
of  Laws  and  Andre wes  (Laws  and  Andre wes,  1894),  who 
entirely  failed  to  isolate  the  typhoid  bacillus  from  the 
sewage  of  London  and  found  only  two  colonies  of  the 
organism  on  a  long  series  of  plates  made  from  the  sewage 
of  a  hospital  containing  forty  typhoid  patients.  So 
Wathelet  (Wathelet,  1895)  found  that  of  600  colonies 
isolated  from  typhoid  stools  and  having  the  appearance 
characteristic  of  B.  coli  and  B.  typhi,  only  10  belonged 
to  the  latter  species. 

In  the  last  five  years  considerable  progress  has  been 
made  in  the  development  of  new  methods  for  isolating 
the  typhoid  bacillus.  These  fall  in  four  distinct  groups: 
first,  the  direct  isolation  by  differential,  frequently  colored, 
media;  second,  enrichment  methods;  third,  methods 
based  on  concentration  of  the  organisms  by  agglutination 
with  typhoid  serum;  and  fourth,  methods  based  on  concen- 
tration by  chemical  precipitation. 

In  all  excepting  the  first  of  these  groups  differential 
media  are  usually  employed  as  a  second  step  in  the  iso- 
lation. Combinations  of  methods  have  been  employed 
in  many  instances,  and  have  often  been  successful  in  the 
isolation  of  the  typhoid  bacillus  from  artificially  infected 
emulsions  of  feces  and  waters. 

Direct  Isolation.  —  Drigalski  and  Conradi  (Drigalski 
and  Conradi,  1902)  prepared  a  medium  primarily  for  the 
isolation  of  typhoid  bacilli  from  excreta,  which  may  also 


7O  Elements  of  Water  Bacteriology. 

be  applied  in  water  bacteriology.  This  consists  of  an 
agar  medium  containing  nutrose,  sodium  chloride,  litmus, 
lactose,  and  a  dye,  "crystal  violet";  and  it  is  used  in  the 
form  of  plate  cultures  infected  by  smearing  the  surface 
after  thorough  cooling  with  a  bent  glass  rod.  The  cul- 
ture medium  is  a  selective  one,  ordinary  saprophytes 
failing  to  grow,  while  after  fourteen  to  twenty-four 
hours  at  37  degrees,  colon  and  typhoid  colonies  can  be 
readily  distinguished  from  one  another.  The  colon  bacil- 
lus produces  red,  non-transparent  colonies,  of  variable 
size  and  depth  of  color,  while  the  typhoid  colonies  are 
blue  or  violet,  transparent,  and  of  smaller  size,  seldom 
exceeding  three  millimeters  in  diameter. 

Endo  (Endo,  1904)  has  suggested  the  use  of  a  fuchsin- 
lactose-agar  decolorized  by  sodium  sulphite.  Upon  this 
medium  B.  coli  produces  bright  red,  sharply  denned, 
round  colonies  in  24  hours  at  37  degrees,  while  B.  typhi 
gives  round,  colorless,  transparent  colonies  with  thin  mar- 
gins. This  medium  has  been  somewhat  modified  by 
Gaehtgens  (Gaehtgens,  1905)  by  the  addition  of  caffeine, 
and  he  found  it  of  great  service  in  isolating  the  typhoid 
bacillus  from  stools  of  patients  suffering  with  the  disease. 
No  attempts  were  made  by  him  to  isolate  the  organism 
from  polluted  water. 

LoefBer  (Loeffler,  1903  and  1906)  and  Lentz  and 
Tietz  (Lentz  and  Tietz,  1903  and  1905)  have  made  use  of 
an  agar  medium  containing  malachite  green.  This 
medium  is  supposed  to  inhibit  the  growth  of  B.  coli 


Isolation  of  Specific  Pathogenes.  71 

while  favoring  B.  typhi,  and  has  been  recommended  for 
the  isolation  of  the  organism  from  feces.  Doebert 
(Dcebert,  1900)  has  shown  that  certain  varieties  of  mala- 
chite green  are  not  suited  to  this  purpose.  Nowack 
(Nowack,  1905)  has  also  pointed  out  the  same  fact,  and 
ascribed  the  difference  to  the  presence  of  dextrin.  He 
finds  that  a  medium  0.8  per  cent  alkaline  to  phenol- 
phthalein  is  more  favorable  to  B.  typhi  and  less  favor- 
able to  B.  coli  than  one  neutral  to  litmus.  With  such 
a  medium  about  20  per  cent  of  the  typhoid  bacilli  present 
develop. 

More  regently  a  considerable  degree  of  success  has 
been  attained  by  methods  based  upon  the  inhibitory  action 
of  caffein  for  B.  coli. 

This  important  fact,  which  was  announced  by  Roth 
(Roth,  1903),  has  given  rise  to  much  investigation,  and 
offers  what  is  probably  the  most  promising  method  for  the 
isolation  of  the  typhoid  bacillus  from  water.  Hoffman 
and  Ficker  (Hoffman  and  Ficker,  1904)  have  developed 
methods  for  the  isolation  of  B.  typhi  from  feces  and 
from  infected  water  by  its  use  in  connection  with  nutrose 
and  crystal  violet.  For  the  isolation  from  infected  water 
solutions  are  prepared  as  follows: 

1.  Ten  grams   of  nutrose  in  80  c.c.  of   sterilized  dis- 
tilled water. 

2.  Five  grams  caffein,   in  20  c,c.   sterilized   distilled 
water. 


72  Elements  of  Water  Bacteriology. 

3.  One- tenth  gram  of  crystal  violet  in  100  c.c.  water. 
Solutions  i  and  2  are  mixed  by  shaking  together  in  a 
flask,  and  the  mixture  poured  into  a  flask  containing  900 
cubic  centimeters  of  the  water  to  be  tested.  10  c.c.  of 
solution  3  are  gradually  added,  and  the  whole  thoroughly 
mixed  by  shaking  and  then  incubated  at  37  degrees  for 
not  over  12-13  hours.  At  the  end  of  the  incubation 
period  loopfuls  of  the  solution  are  smeared  over  Drigal- 
ski-Conradi  plates. 

By  this  method  the  B.  typhi  was  isolated  from  mixtures 
in  river  water  containing  one  typhoid  bacillus  to  51,867 
water  bacteria  and  colon  bacilli. 

A  number  of  investigations  have  shown  that  the  action 
of  the  caffein  is  not  as  markedly  selective  as  at  first 
claimed.  Kloumann  (Kloumann,  1904)  obtained  no 
better  results  by  this  method  than  by  the  Drigalski- 
Conradi  medium  alone,  and  Willson  (Willson,  1905) 
found  that  certain  strains  of  B.  typhi  were  inhibited, 
while  strains  of  B.  coli  developed  feebly  in  the  presence 
of  0.5  per  cent  of  caffein. 

The  phenomenon  of  agglutination  was  made  the  basis 
of  a  method  of  isolating  B.  typhi  from  water  by  Adami 
and  Chopin  (Adami  and  Chopin,  1904).  Two-liter 
samples  of  the  water  were  collected  in  sterilized  bottles 
(Winchester  quarts),  and  to  each  was  added  twenty  cubic 
centimeters  of  one  per  cent  glucose  broth.  The  sample 
was  incubated  for  18  to  24  hours  at  37°  C.,  after 
which  ten  cubic  centimeter  portions  were  withdrawn  and 


Isolation  of  Specific  Pathogenes.  73 

placed  in  long,  narrow  test  tubes.  To  each  of  these  tubes 
enough  typhoid  serum  of  known  potency  was  added  to 
make  a  regularly  graded  series,  1-50,  i-ioo,  1-150, 
and  1-200.  The  probable  presence  of  the  typhoid 
bacillus  was  manifest  by  the  formation  of  flocculi  within 
a  quarter  of  an  hour,  and  agglutination  was  complete  in 
from  two  to  five  hours. 

The  tube  having  the  greatest  dilution  in  which  aggluti- 
nation was  apparent  was  then  examined  by  breaking  off 
the  lower  end,  containing  the  precipitate,  washing  the 
sediment  two  or  three  times  with  sterile  water  after 
removing  the  clear  supernatant  liquid,  and  allowing  the 
bacteria  to  settle  again.  The  organisms  remaining  were 
plated  upon  various  media,  and  examined  biochemically 
to  determine  their  true  character.  It  was  found  that  a 
dilution  of  i  to  60  was  the  highest  which  could  be  used 
with  the  organisms  examined,  and  it  is  therefore  prob- 
able that  high  dilutions  (greater  than  1-60)  cannot  be 
successfully  used. 

A  study  of  the  organisms  isolated  in  this  case  was 
made  by  Klotz  (1904),  who  found  the  culture  to  be 
not  a  typical  B.  typhi  but  a  form  showing  certain 
points  of  similarity  to  both  B.  typhi  and  to  B.  coli,  and 
probably  intermediate  between  them.  As  this  author 
pointed  out,  it  is  therefore  evident  that  even  when  a 
positive  result  is  obtained  with  a  relatively  high  dilution 
of  typhoid  serum  it  is  unwise  to  regard  the  action  as 
absolutely  specific. 


74  Elements  of  Water  Bacteriology. 

Schepilewski  (Schepilewski,  1903)  and  Altschuler 
(Altschuler,  1903)  have  also  used  agglutination  as  a  means 
of  precipitating  the  bacteria  after  enrichment  cultivation 
in  broth.  The  former  incubated  the  culture  at  37  degrees 
for  24  hours,  then  added  a  serum  of  high  potency,  allowed 
the  mixture  to  stand  for  two  to  three  hours  and  then 
centrifuged.  The  supernatant  liquid  was  removed,  and 
the  mass  of  agglutinated  cells  broken  up  by  shaking 
with  glass  beads  and1  salt  solution.  Upon  plating  upon 
litmus  lactose  agar  the  organisms  could  be  detected. 
In  this  way  positive  isolation  was  made  from  water 
containing  i  loopful  of  a  broth  culture  in  50  liters  of 
water.  Altschuler 's  method  of  enrichment  was  essen- 
tially like  that  of  Schepilewski.  From  the  surface  of  the 
culture  developed  at  37  degrees,  10  c.c.  were  removed 
to  a  tapering  tube  provided  with  a  rubber  tube  at  the 
bottom.  Serum  was  added  in  the  proportion  of  one  part 
in  50,  the  culture  agitated  to  release  entangled  non- 
agglutinated  bacilli  and  the  sediment  run  into  a  tube 
containing  i  per  cent  peptone  and  J  per  cent  salt.  The 
agglutinated  mass  was  broken  up  by  shaking  with  sand, 
and  the  culture  incubated  at  37  degrees  for  24  hours,  and 
then  plated  on  Drigalski-Conradi  plates.  The  organism 
was  isolated  from  dilute  suspensions  in  water  (150  in 
i  liter)  and  also  from  the  feces  of  a  typhoid  patient  with 
which  other  methods  gave  negative  results. 

A  number  of  methods  for  concentrating  typhoid  bacilli 
in  water  by  chemical  precipitation  have  been  tested  experi- 


Isolation  of  Specific  Pathogenes.  75 

mentally,  with  some  degree  of  promise.  Vallet  (Vallet, 
1901)  was  the  first  to  employ  this  principle,  and  made 
use  of  sodium  hyposulphite  and  lead  acetate.  The  mix- 
ture was  centrifugalized  and  the  precipitate  dissolved 
in  more  hyposulphite.  The  clear  solution  was  then 
plated. 

Schiider  (Schuder,  1903)  observed  that  the  lead  salt 
reacted  harmfully  upon  the  bacteria,  and  pointed  out 
that  the  hypolsuphite  should  be  in  excess.  In  his  ex- 
periments water  was  allowed  to  stand  in  tall  jars  for  24 
hours.  To  2  liters  of  infected  water,  20  c.c.  of  a  7.75  per 
cent  solution  of  sodium  hyposulphite  was  added,  and 
after  thorough  mixing  20  c.c.  of  a  10  per  cent  solution  of 
lead  nitrate.  The  precipitate,  after  20  to  24  hours,  was 
treated  with  14  c.c.  of  saturated  sodium  hyposulphite 
solution  and  shaken.  From  the  clear  solution  0.2  to 
0.5  c.c.  portions  were  streaked  upon  Drigalski-Conradi 
plates  which  were  then  incubated  at  37  degrees  for  24 
hours.  Ficker  (Ficker,  1904)  modified  the  process  still 
more  by  using  ferric  sulphate,  and  dissolved  the  precipi- 
tate with  neutral  potassium  tartrate.  The  final  solution 
was  then  plated  on  Drigalski-Conradi  medium.  Ficker 
claimed  that  this  method  gives  excellent  results,  97-98 
per  cent  of  the  typhoid  bacteria  being  carried  down  with 
the  precipitate. 

Muller  (Miiller,  1905),  after  comparing  different  precipi- 
tation methods,  adopted  ferric  oxychloride  as  the  most 
suitable  precipitant,  because  of  its  quicker  and  less  de- 


76  Elements  of  Water  Bacteriology. 

structive  action.  Willson  (Willson,  1905)  suggested  the 
use  of  alum  as  a  precipitant.  He  added  0.5  gr.  alum  per 
liter  of  water  examined.  The  mixture  was  then  cen-- 
trifugalized,  and  the  precipitate  suspended  in  a  small 
amount  of  water  and  plated  on  Drigalski-Conradi  medium. 
Nieter  (Nieter,  1906)  made  20  parallel  experiments,  using 
very  pure  water  infected  with  typhoid  bacilli  in  varying 
numbers.  By  precipitating  with  ferric  sulphate  and  sodium 
hydrate,  centrifugalizing,  and  then  filtering  through  a 
sterile  filter  he  obtained  results  with  small  numbers  of 
bacteria.  Using  iron  oxychloride  as  the  precipitant,  he 
confirmed  the  results  of  Miiller.  By  plating  on  mala- 
chite-green agar  he  was  often  able  to  get  positive  results 
when  the  Drigalski-Conradi  medium  failed. 

By  use  of  a  combination  of  enrichment  and  chemical 
precipitation,  Ditthorn  and  Gildemeister  (Ditthorn  and 
Gildemeister,  1906)  isolated  the  typhoid  bacillus  from 
enormous  artificial  dilutions  in  water.  In  the  typhoid 
fever  epidemic  in  Posen,  in  1906,  it  was  found  that  the 
bile  of  those  dying  from  the  disease  contained  nearly  pure 
cultures  of  typhoid  bacilli.  This  led  the  authors  mentioned 
to  use  bile  and  bile  agar  as  enrichment  media.  After 
precipitating  by  Miiller's  method,  the  whole  of  the  pre- 
cipitate was  added  to  100  c.c.  sterile  ox  bile  and  grown 
at  37  degrees  for  24  hours,  after  which  time  i  c.c.  portions 
were  plated.  With  extreme  dilutions  it  was  found  desir- 
able to  incubate  for  48  to  72  hours.  The  results  were 
unsatisfactory  in  the  presence  of  large  numbers  of  water 


Isolation  of  Specific  Pathogenes. 


77 


bacteria.  It  is  also  pointed  out  that  the  iron  oxychloride 
is  bactericidal  in  48  hours. 

Drigalski  (Drigalski,  1906)  has  suggested  the  separa- 
tion of  B.  typhi  from  other  bacteria  in  water  through 
its  greater  motility.  He  succeeded  in  isolating  typhoid 
bacilli  from  two  springs  by  the  following  method:  five 
to  ten  liters  of  water  were  allowed  to  stand  for  one  to  two 
days  in  tall  milk  cans  at  room  temperature.  Samples 
were  taken  from  the  surface  and  plated  on  litmus  lactose 
agar  (Drigalski-Conradi  medium),  the  amount  of  water 
to  be  used  varying  with  the  contamination. 

The  most  promising  methods  for  examination  of  water 
for  B.  typhi  may  be  conveniently  summarized  in  the 
following  tabular  view  adapted  from  Willson's  paper. 

TABULAR   SUMMARY. 


Examination 

of  water 

for  Ty-      - 

phoid 

bacilli. 


Isolation 


(1)  Filtration. 

(2)  Chemical  precip. 


f  Schiider's  process. 
I  Fkker's  process. 
I  Alum  process. 
L  Muller's  process. 

(3)  Serum  agglutination. 

(4)  Enrichment— Hoffman  and  Picker's  process. 

(5)  Cambier's  process. 

Gelatin  (Eisner's,  etc.) 

Bile-salt  agar. 

Glucose  and  lactose  agars. 


(6)  Solid  media 


Drigalski-Conradi  medium. 
Endo's  medium 
Loeffler's      malachite-green 
agar. 


(Morphological  and  cultural  characters,  etc. 
Identification  (Agglutination. 

I  r  XPfeiffer's,  etc. 

Of  the  comparative  advantages  of  these  methods  it  is 
still  too  early  to  speak  with  finality.     Up  to  the  present 


7  8  Elements  of  Water  Bacteriology. 

time  the  use  of  caffein  has  apparently  been  followed  by 
the  best  results,  and  it  seems  likely  that  of  the  precipita- 
tion methods  that  employing  the  oxychloride  of  iron  is  the 
best.  Lubenau  (Lubenau,  1907)  has  made  some  inter- 
esting comparisons,  using  media  containing  malachite 
green  and  caffein  and  caffein  alone,  in  which  the  advan- 
tage is  decidedly  in  favor  of  the  latter. 

After  this  part  of  the  process  is  completed  the  identifi- 
cation of  the  pure  cultures  isolated  is  subject  to  consider- 
able uncertainty.  The  typhoid  bacillus  belongs  to  a  large 
group  which  contains  numerous  varieties  differing  from 
each  other  by  minute  degrees.  The  inability  to  repro- 
duce the  disease  by  inoculation  in  available  test  animals, 
owing  to  their  natural  immunity,  is  a  serious  drawback; 
and  the  specific  biochemical  characters  of  the  organism 
are,  as  it  happens,  mostly  negative  ones,  as  shown  by  com- 
parison with  B.  coli,  to  which  it  is  supposed  to  be  allied. 

COMPARISON    OF    THE    CHARACTERS    OF    B.   COLI   AND 
B.   TYPHI. 

(HORROCKS,  1901.) 
B.  coli.  B.  typhi. 

(1)  Surface     Colonies,     Gelatin          (i)  Much    thinner    than    those 
Plates.  —  Thicker,  and  grow  more  of  B.  coli,  and  grow  more  slowly, 
rapidly    than    those    of   B.    typhi.  After    forty-eight    hours'    incuba- 
After  forty -eight  hours'  incubation  tion    at    22°  C.    they    are    hardly 
at   22°  C.    they   are   usually  large  visible  to  the  naked  eye. 

and  characteristic. 

(2)  Gelatin-stab. — Quick  growth  (2)  Slow  growth  on  the  surface 
on  the  surface  and  along  the  line  like  the  colonies;  along  the  line  of 
of  inoculation.  inoculation    the   growth   is   much 

thinner,  and  often  ends  below  in  a 
few  white  points  consisting  of  dis- 
crete colonies. 


Isolation  of  Specific  Pathogenes. 


79 


B.  coli. 

(3)  Gelatin-slope. — Thick,  broad, 
grayish-white  growth  with   a  cre- 
nated  margin. 

(4)  Witte's    Peptone    and    Salt 
Solution.  —  Indol  produced. 

(5)  Milk.  —  Coagulated. 

(6)  Litmus- whey,  one  week  at 
37°  C.     Acid     produced,     usually 
requiring  from  20  to  40  per  cent  of 

^T 

—  alkali  to  neutralize  it. 
10 

(7)  Neutral-red  Glucose-agar. — 
Marked  green  fluorescence. 

(8)  Glucose-gelatin     and     Lac- 
tose-gelatin   Shake   Cultures,    and 
Glucose-agar-stab.  —  Marked  gas 
formation. 

(9)  Gelatin,  25  per  cent,  incu- 
bated    at     37°  C.'  —  Thick    -film 
appears  on  the  surface. 

(10)  Potato. —  As  a  rule,  a  thick 
yellowish-brown  growth. 

(n)  Proskauer  and  Capaldi's 
Media.  No.  I,  after  twenty  hours 
growth,  medium  acid.  No.  II, 
Growth,  medium  neutral  or  faintly 
alkaline. 

(12)  Nitrate-broth. — Nitrate  re- 
duced to  nitrite. 

(13)  Microscopical         Appear- 
ances. —  A    small    bacillus    often 
like  a  coccus,  not  motile  as  a  rule. 

(14)  Flagella.  —  Usually  i  to  3, 
short  and  brittle;  sometimes  8  to 
12,  long  and  wavy. 

(15)  Agglutination.  —  As  a  rule, 
no  agglutination  with  a  dilute  anti- 
typhoid serum. 


B.  typhi. 

(3)  Thin,  narrow,  grayish-white 
growth,     crenated      margin     not 
marked  as  in  B.  coli. 

(4)  No  formation  of  indol. 

(5)  Unchanged  after  a  month. 

(6)  Very  small  amount  of  acid 
produced,  requiring  not  more  than 

N 

6  per  cent  of  —  alkali  to  neutral- 
10 

ize  it. 

(7)  No  change. 

(8)  No  gas  formation. 


(9)  No  film  appears  on  the  sur- 
face, but  a  general  growth  takes 
place  throughout  the  tube. 

(10)  Thin    transparent    growth 
hardly  visible  to  the  naked  eye. 

(n)  No.  I,  no  growth  or  change 
in  the  reaction  of  the  medium. 
No.  II,  Growth,  medium  acid. 


(12)  Reduction  of  nitrate  not  so 
marked. 

(13)  Usually    longer    than    B. 
coli;    highly  motile,  with  a  quick 
serpent-like  movement. 

(14)  Usually  8  to  12,  long  and 
wavy. 

(15)  Marked  agglutination  with 
dilute  anti-typhoid  serum. 


Of  the  many  observers  who  have  reported  the  isola- 
tion of  the  typhoid  bacillus  from  water,  all  but  the  most 
recent  are  quite  discredited,  on  account  of  the  insufficiency 
of  their  confirmatory  tests;  and  even  the  latest  results 


8o  Elements  of  Water  Bacteriology. 

should  be  received  with  caution.  Since  the  introduction 
of  the  Widal  (Widal,  1896)  reaction,  founded  on  the 
fact  that  typhoid  bacilli  examined  under  the  microscope 
in  the  diluted  blood-serum  of  a  typhoid  patient  lose  their 
motility  and  "  agglutinate "  or  clump  together,  an  impor- 
tant aid  has  been  furnished  in  the  diagnosis.  Yet  serum 
tests  are  notably  erratic,  and  insufficient  to  identify  an 
organism  without  an  exhaustive  study  of  biochemical 
reactions.  Many  organisms  are  agglutinated  by  typhoid 
serum  in  a  more  or  less  dilute  solution,  and  agglutina- 
tion tests  are  not  significant  unless  obtained  in  dilutions 
as  great  as  1-500  or  i-iooo.  The  discovery  of  the 
Bacillus  dysenteriae  of  Shiga,1  which  closely  resembles 
the  typhoid  bacillus,  has  made  the  identification  of  the 
latter  more  dubious  than  ever.  Hiss  (1904)  has  shown 
that  the  fermentation  and  agglutination  reactions  of  the 
two  organisms  are  in  many  respects  alike,  and  Park  and 
his  associates  (1904)  have  shown  that  there  are  not  less 
than  three  distinct  types  of  dysentery  bacilli  forming  that 
group. 

In  the  work  so  far  described  the  typhoid  organism  was 
not  isolated  from  polluted  water,  but  from  artificial 
mixtures  or  excreta.  There  are,  however,  a  number  of 
cases  in  which  the  organism  has  undoubtedly  been  iso- 
lated from  polluted  water,  as  by  Kiibler  and  Neufeld 
(Kiibler  and  Neufeld,  1899),  who  examined  a  farmhouse 

1  For  an  account  of  the  Biology  of  B.  dysenteriae  the  student  is 
referred  to  an  article  by  Dombrowsky,  1903. 


Isolation  of  Specific  Pathogenes.  8 1 

well  at  Neumark  in  1899,  and  Fischer  and  Flatau 
(Fischer  and  Flatau,  1901),  who  discovered  an  organ- 
ism responding  to  a  most  exhaustive  series  of  tests  for 
the  typhoid  bacillus  in  a  well  at  Rellingen  in  1901.  In 
these  cases  the  water  was  directly  plated  upon  Eisner's 
medium  or  phenolated  gelatin  with  no  preliminary  process 
of  enrichment.  Willson  (Willson,  1905)  has  summarized 
the  instances  in  which  the  typhoid  bacillus  has  been  iso- 
lated from  infected  drinking  water,  and  includes,  in  addi- 
tion to  the  above-mentioned  cases,  the  following: 

1.  By  Losener,  in  1895,  from  the  Berlin  water  supply. 

2.  By  Conradi,  in  1902,  from  a  well  at  Pecs  in  Hun- 
gary, by  use  of  carbol  gelatin  plates.    . 

3.  By  Jaksch  and  Rau,  in  1904,  from  the  water  supply 
of  Prague,  and  also  from  the  river  Moldau,  by  caffeine- 
nutrose  crystal  violet  agar. 

4.  By  Stroszner,  in  1904,  from  a  well  near  Budapest, 
by  the  same  method. 

The  search  for  the  typhoid  bacillus  is  usually  suggested 
when  an  outbreak  of  the  disease  has  cast  strong  suspicion 
upon  some  definite  source  of  water-supply.  By  the  time 
an  epidemic  manifests  itself,  however,  the  period  of  the 
original  infection  is  long  past,  and  the  chances  are  good 
that  any  of  the  specific  bacilli  once  present  will  have  dis- 
appeared. While  elaborate  experiments  have  shown  that 
B.  typhi  may  persist  in  sterilized  water  for  upwards  of 
two  months  and  in  unsterilized  water  from  three  days 
to  several  weeks,  the  number  of  the  organisms  present  is 


82  Elements  of  Water  Bacteriology. 

always  very  rapidly  reduced  (Frankland,  1894).  More 
recently,  Jordan  (Jordan,  1905)  has  demonstrated  that 
the  typhoid  bacillus  may  be  isolated  from  mixed  cultures 
of  B.  typhi  and  B.  coli  in  tap  water  and  sewage  after 
thirty-four  days,  with  unchanged  agglutinative  powers. 
On  the  other  hand,  Jordan,  Russell  and  Zeit  (Jordan, 
Russell  and  Zeit,  1904),  and  Russell  and  Fuller  (Russell 
and  Fuller,  1906)  have  shown  that  in  unsterilized  lake 
water,  river  water,  and  sewage,  the  life  of  the  organism 
may  not  exceed  five  days.  Whipple  and  Mayer  (Whipple 
and  Mayer,  1906)  have  ascribed  to  dissolved  oxygen  a 
decided  effect  upon  the  viability  of  the  typhoid  bacillus 
in  water,  absence  of  oxygen  tending  to  weaken  the 
organism. 

Epidemiological  evidence  confirms  the  results  of  Laws 
and  Andrewes  which  teach  that  the  number  of  typhoid 
bacilli  even  in  polluted  water  is  probably  never  very  great, 
while  the  fate  of  Lowell  and  Lawrence  in  1890-91  and 
the  more  recent  epidemics  at  Butler,  Pa.,  and  Ithaca,  N.  Y., 
seem  strongly  to  demonstrate  that  even  a  small  number 
of  virulent  organisms  can  bring  about  an  almost  wholesale 
infection.  Indeed,  if  the  virulent  organism  were  as 
abundant  as  some  results  would  indicate  (Remlinger 
and  Schneider,  1897),  the  human  race  would  long  since 
have  been  exterminated.  On  the  whole  it  is  clear  that  a 
negative  test  for  the  typhoid  bacillus  means  practically 
nothing.  Since  this  is  so,  and  since  a  positive  result  is 
always  open  to  serious  doubt,  the  search  for  the  typhoid 


Isolation  of  Specific  Pathogenes.  83 

bacillus,  however  desirable  theoretically,  cannot  be  re- 
garded at  present  as  generally  profitable. 

The  isolation  of  the  cholera  bacillus  from  water  can 
probably  be  accomplished  with  somewhat  less  difficulty 
than  is  encountered  in  the  case  of  B.  typhi.  Schottelius 
(Schottelius,  1885)  was  the  first  to  point  out  the  necessity 
for  growing  this  organism  in  an  alkaline  medium,  and 
Loeffier  (Loeffler,  1893)  found  that  its  isolation  from 
water  could  be  successfully  accomplished  by  adding  10  c.c. 
of  alkaline  peptone  broth  to  200  c.c.  of  the  infected  water 
and  incubating  for  twenty-four  hours  at  37  degrees,  when 
the  organism  could  be  found  at  the  surface  of  the  medium. 

Somewhat  earlier  than  this  Dunham  (Dunham,  1887) 
had  made  a  special  study  of  the  chemical  reactions  of  the 
cholera  bacillus  and  found  that  the  organism  would  grow 
abundantly  in  a  solution  containing  i  per  cent  peptone 
and  .5  per  cent  salt  (Dunham's  solution),  producing  the 
"  cholera-red  or  nitroso-indol  reaction."  This  medium 
was  brought  into  practical  use  by  Dunbar  (Dunbar,  1892), 
who  succeeded  in  isolating  the  organisms  from  the  water 
of  the  Elbe  in  1892,  during  the  cholera  epidemic  at 
Hamburg. 

Koch  (Koch,  1893)  prescribed  the  following  method  for 
the  isolation  of  the  organism  from  water: 

To  100  c.c.  of  the  water  to  be  examined  is  added  i 
per  cent  peptone  and  i  per  cent  salt.  The  mixture  is 
then  incubated  at  37  degrees.  After  intervals  of  ten, 
fifteen,  and  twenty  hours,  the  solution  is  examined  micro- 


84  Elements  of  Water  Bacteriology. 

scopically  for  comma-shaped  organisms,  and  agar  plate 
cultures  are  made  which  are  likewise  incubated  at  37 
degrees.  If  any  colonies  showing  the  characteristic* 
appearance  of  the  cholera  bacillus  are  found,  these  are 
examined  microscopically,  and  if  comma-shaped  organ- 
isms are  present,  inoculations  are  made  into  fresh  tubes 
to  be  further  tested  by  means  of  the  indol  reaction  and  by 
inoculation  into  animals.  The  existence  of  other  spirilla 
of  some  pathogenic  power  renders  necessary  the  greatest 
care  and  caution  in  claiming  positive  isolations.  That 
no  great  improvement  on  Koch's  method  has  been  made 
during  the  last  ten  years  seems  apparent  from  the  state- 
ments of  Kolle  and  Gotschlich  (Kolle  and  Gotschlich, 
1903),  who  employed  "  the  peptone  method  with  subse- 
quent agar  cultivation  "  in  the  isolation  of  the  organisms 
from  feces  of  cholera  patients  during  the  epidemic  in 
Egypt  in  1902. 

Other  pathogenic  organisms  have  been  isolated  from 
waters,  according  to  the  accounts  of  numerous  investi- 
gators, but  from  the  sanitary  point  of  view  the  typhoid 
and  cholera  bacilli  are  of  most  importance  since  these  are 
manifestly  the  germs  of  disease  most  likely  to  be  dissemi- 
nated through  this  medium.  For  the  detection  of  B. 
anthracis  and  other  spore-forming  pathogenic  bacteria 
which  may  at  times  gain  access  to  water  from  stockyards, 
slaughter-houses,  etc.,  the  method  suggested  by  Frankland 
(Frankland,  1894)  may  be  adopted.  The  water  to  be 
examined  is  heated  to  90  degrees  for  two  minutes  and  then 


Isolation  of  Specific  Pathogenes.  85 

plated,  the  characteristic  colonies  of  the  anthrax  organism 
being  much  more  easily  discerned  after  the  destruction  of 
the  numerous  non-sporing  water  bacteria.  Again,  water 
is  sometimes  the  means  of  distributing  the  germs  of 
dysentery  and  diarrhoea,  as  shown  by  the  decrease  of  these 
diseases  in  Burlington,  Vt.  (Sedgwick,  1902),  and  other 
communities  where  pure  water-supplies  have  been  sub- 
stituted for  polluted  ones.  Thresh  (Thresh,  1903) 
described  an  epidemic  of  over  1000  cases  of  diarrhoea  with 
14  deaths,  which  occurred  in  England  at  Chelmsford  and 
Widford,  and  was  undoubtedly  spread  by  the  public 
water-suppK  A  somewhat  similar  epidemic  of  dysentery 
occurred  in  Warren  and  Kittanning,  in  Pennsylvania,  in 
1906,  as  a  result  of  contamination  of  the  water,  in  this 
case  a  river-supply.  It  is  possible  that  the  examination  of 
water  for  the  B.  dysenteriae  may  in  the  future  help  to 
throw  important  light  on  its  sanitary  condition. 


CHAPTER  VI. 

» 
METHODS  FOR  THE  ISOLATION  OF  THE  COLON  BACILLUS. 

THE  Bacillus  coli  was  first  isolated  by  Escherich 
(Escherich,  1884)  from  the  feces  of  a  cholera  patient. 
It  was  subsequently  found  to  be  a  normal  inhabitant  of 
the  intestinal  tract  of  man  and  many  other  animals,  and 
to  occur  regularly  in  their  excreta,  and  on  this  account 
it  became  of  the  highest  interest  and  importance  to  sani- 
tarians, since  its  presence  in  water-supplies  was  regarded 
as  direct  evidence  of  sewage  pollution. 

This  organism  may  be  described  as  a  short,  usually 
motile  rod,  with  diameter  generally  less  than  one  micron 
and  exhibiting  no  spore  formation.  It  forms  thin  irregu- 
lar translucent  films  upon  the  surface  of  gelatin,  called 
" grape-leaf  colonies"  by  the  Germans,  produces  no 
liquefaction,  and  gives  a  wire-nail-like  growth  in  stick 
cultures.  It  forms  a  white  translucent  layer  of  character- 
istic appearance  upon  agar,  produces  a  more  or'  less 
abundant,  moist,  yellowish  growth  on  potato,  and  tur- 
bidity and  some  sediment  in  broth;  it  ferments  dextrose 
and  lactose  with  the  formation  of  gas  of  which  the  ratio  is 

approximately,   -  —    =   —   as   ordinarily   determined;   a 
CO2          i, 

86 


Isolation  of  the  Colon  Bacillus.  87 

strong  acid  reaction  is  developed  in  most  sugar-containing 
media.  The  organism  reduces  nitrates  to  nitrites  and 
sometimes  to  ammonia.  It  reduces  neutral  red,  changing 
its  color  to  canary  yellow  with  a  greenish  fluorescence.  It 
grows  in  the  Capaldi-Proskauer  media,  forming  acid  in  the 
albumin-free  medium,  No.  i,  and  giving  a  neutral  or 
alkaline  reaction  in  the  pepton-mannite  medium,  No.  2. 
It  coagulates  casein  in  litmus  milk,  and  reduces  the  litmus 
with  subsequent  slow  return  of  the  color  (red),  and  forms 
indol  in  pepton  solution.  Many  cultures  of  this  organism 
are  fatal  to  guinea  pigs  when  the  latter  are  inoculated 
subcutaneously  with  one-half  c.c.  of  a  twenty-four-hour 
bouillon  culture,  and  most  cultures  produce  death  when 
this  amount  is  inoculated  intraperitoneally.  Although 
not  a  spore-forming  bacillus,  and  in  general  not  possessing 
great  resistance  against  antiseptic  substances,  B.  coli  is 
less  susceptible  to  phenol  than  are  many  other  forms, 
especially  certain  water-bacteria. 

A  word  may  be  added  as  to  the  fermentative  powers  of 
the  colon  group  in  other  carbohydrates  than  dextrose  and 
lactose.  Of  the  monosaccharides,  galactose,  like  dextrose, 
is  always  fermented;  and  among  the  polysaccharides, 
maltose  and  xylose  are  broken  up  as  well  as  lactose. 
Inulin  is  not  attacked.  The  alcohols,  mannite  and  dulcite, 
are  fermented  by  some  strains  and  not  by  others.  The 
colon  group,  as  Smith  (1893)  long  ago  pointed  out,  may 
be  divided  into  two  distinct  subtypes  according  to  the 
action  of  the  organisms  upon  saccharose.  One  subtype 


88  Elements  of  Water  Bacteriology. 

forms  gas  and  acid  in  saccharose  media  and  the  other  does 
not.  Winslow  and  Walker  (1907)  have  recently  found 
that  those  strains  which  ferment  saccharose  attack  raffinose 
also,  and  point  out  that  these  two  sugars  which  behave 
alike  are  those  which  lack  the  aldehyde  grouping  char- 
acteristic of  dextrose  and  lactose.  The  name  B.  coli 
communior  was  given  to  the  saccharose  fermenting  type 
by  Durham  (1901);  as  Ford  (1903)  suggests,  this  name 
should  be  changed  to  B.  communior  in  deference  to  the 
established  binomial  rule  of  biological  nomenclature. 
The  term,  B.  coli,  should,  in  strictness,  apply  only  to  those 
forms  which  fail  to  attack  the  ketonic  sugars.  For 
practical  sanitary  purposes,  however,  the  distinction  is 
unimportant.  Throughout  this  book,  therefore,  both  B. 
communior  and  B.  coli,  proper,  will  be  considered  together 
under  the  name  of  the  colon  bacillus,  which  is  almost 
universally  applied  to  both. 

The  litmus-lactose-agar  plate  (Wurtz,  1892),  as  noted 
in  Chapter  IV,  furnishes  one  ready  method  for  the  iso- 
lation of  B.  coli  from  water,  and  it  was  used  by  Sedg- 
wick  and  Mathews  for  the  purpose  as  early  as  1893 
(Mathews,  1893).  The  process  is  based  upon  the  fact 
already  alluded  to,  that  B.  coli  readily  ferments  lactose 
with  the  formation  of  acid.  If,  therefore,  plates  are  made 
with  agar  containing  both  lactose  and  litmus,  the  colon 
colonies  develop  as  red  spots  in  a  blue  field.  Since 
organisms  other  than  B.  coli  may  also  develop  red  colonies, 
it  is  necessary  to  examine  the  red  colonies  further.  This 


Isolation  of  the  Colon  Bacillus.  89 

is  done  by  fishing  from  isolated  colonies,  replating  and 
inoculating  into  the  usual  media  for  identification. 

The  plate  method  of  isolation  is  recommended  by  the 
Committee  on  Standard  Methods  of  Water  Analysis 
(1905)  for  sewages  and  polluted  waters,  and  with  such 
sources  it  yields  good  results.  For  success  in  the  use  of 
this  method  it  is  necessary  to  get  a  sufficient  dilution  so 
that  colonies  may  be  well  isolated,  and  to  this  end  it  is 
advisable  that  a  number  of  different  dilutions  be  employed, 
a  series  of  plates  being  prepared  from  each.  Under  any 
conditions  the  detection  of  the  colon  bacillus  is  seriously 
hampered  by  the  development  of  other  forms.  Certain 
observers  have  therefore  added  phenol  to  the  agar  medium, 
combining  the  effect  of  high  temperature  and  an  antiseptic 
to  check  the  growth  of  water-bacteria.  Copeland  for 
this  purpose  added  to  his  tubes  .2  c.c.  of  a  2  per  cent  solu- 
tion of  phenol  (Copeland,  1901).  Chick  (Chick,  1900) 
found  that  1.33  parts  of  phenol  in  1000  materially  de- 
creased the  number  of  colon  bacilli  which  would  develop, 
while  i  part  gave  very  satisfactory  results,  the  plates 
showing  pure  cultures  of  B.  coli.  The  addition  of  anti- 
septics in  this  way  is  always  open  to  the  objection  that 
weaker  strains  may  be  killed  and  lost. 

The  test  for  the  colon  bacillus  in  less  heavily  polluted 
waters  may  be  made  more  delicate  by  a  preliminary 
enrichment  of  the  sample  by  growth  in  a  liquid  medium 
for  twenty-four  hours  at  37  degrees,  thus  greatly  increasing 
the  proportion  of  these  organs  present  before  plating.  As 


90  Elements  of  Water  Bacteriology. 

suggested  in  the  classic  researches  of  Theobald  Smith 
(Smith,  1892),  this  method  may  be  made  approximately, 
quantitative  by  the  inoculation  of  a  series  of  tubes  with 
measured  portions  of  the  water.  If,  for  example,  of  ten 
tubes  inoculated  each  with  T^o  of  a  cubic  centimeter, 
four  show  B.  coli,  we  may  assume  that  some  40  of  these 
organisms  were  present  to  the  cubic  centimeter.  Irons 
(Irons,  1901),  in  a  comparative  study  of  various  methods 
for  the  isolation  of  B.  coli,  showed  that  the  preliminary 
enrichment  frequently  gave  positive  results  when  the 
results  of  the  direct  use  of  the  agar  plate  were  negative, 
and  concluded  that  "  where  the  amount  of  B.  coli  is 
small  and  the  colony  count  large,  the  lactose  plate  for 
plating  water  direct  is  inferior  to  the  dextrose  fermen^ 
tation-tube."  Gage  obtained  similar  results  (Gage,  1902). 

The  medium  ordinarily  used  for  the  preliminary  enrich- 
ment is  ordinary  broth  to  which  i.o  per  cent  of  dextrose 
has  been  added,  and  the  reaction  brought  to  the  neutral 
point.  Into  each  of  a  number  of  fermentation-tubes  of 
this  medium  a  measured  quantity  of  the  water  to  be 
examined  is  inoculated,  and  the  culture  is  incubated  for 
twenty-four  hours  at  37.5°  C.  At  the  end  of  this  time  the 
tubes  are  examined  for  gas  formation.  If  gas  is  found,  a 
small  amount  of  the  culture  should  be  added,  after  suit- 
able dilution,  to  litmus  lactose  agar  and  plated. 

With  polluted  waters  it  will  be  found  advantageous  to 
plate  out  on  the  first  appearance  of  gas  (4-8  hours).  It 
has  been  shown  by  one  of  us  (Prescott,  1902^  that  a  very 


Isolation  of  the  Colon  Bacillus.  91 

rapid  development  of  B.  coli  takes  place  in  the  first  few 
hours  after  dextrose  solutions  are  inoculated  with  intes- 
tinal material,  and  a  nearly  pure  growth  of  colon  bacilli 
often  results,  while  other  bacteria  multiply  more  slowly. 
With  highly  polluted  waters  gas  formation  will  probably 
begin  within  twelve  hours,  but  with  fewer  colon  bacilli 
present  the  duration  must  be  increased.  If  the  period 
of  incubation  be  too  long  continued,  trouble  in  the  subse- 
quent steps  of  the  isolation  may  be  encountered  because 
of  the  overgrowth  of  B.  coli  by  the  sewage  streptococci, 
or  other  forms  which  check  the  growth  of  the  colon  bacilli 
in  the  later  stages  of  fermentation  and  finally  kill  them 
out. 

As  has  already  been  stated,  phenol  has  less  inhibitory 
action  upon  B.  coli  than  upon  normal  water-bacteria, 
and  it  was  hoped  that  a  broth  containing  this  substance 
might  be  employed  for  preliminary  enrichment  with  ad- 
vantage, its  inhibitory  power  checking  the  overgrowing 
forms,  but  not  B.  coli.  This  medium  was  used  in  place 
of  dextrose  broth  for  many  of  the  studies  made  in  con- 
nection with  the  Chicago  drainage  canal  (Reynolds,  1902). 
Phenol  broth  consists  of  ordinary  broth  to  which  o.i  per 
cent  phenol  is  added,  and  the  method  of  procedure  is  to 
add  i  c.c.  of  the  water  to  10  c.c.  of  the  sterilized  phenol 
broth  and  incubate  at  body  temperature  for  twenty-four 
hours.  Litmus-lactose-agar  plates  are  then  made  and 
the  examination  of  the  red  colonies  carried  out  as  described 
for  the  dextrose-broth  method.  It  has  unfortunately 


92 


Elements  of  Water  Bacteriology. 


proved,  however,  that  with  waters  of  fairly  good  quality 
the  phenol  interferes  with  the  colon  bacilli  themselves  to 
a  serious  extent.  The  dextrose  broth  furnishes  a  more 
delicate  test  than  the  carbol  broth  when  the  number  of 
colon  bacilli  present  is  small,  as  is  clearly  shown  by  the- 
following  table  from  Irons : 

PROPORTION  OF  POSITIVE  RESULTS  IN  TESTS  OF 
POLLUTED  AND  UNPOLLUTED  WATERS  BY  DEX- 
TROSE FERMENTATION-TUBE  AND  CARBOL-BROTH 
METHODS. 

(IRONS,   1901.) 


Dextrose 
Fermentation- 
tube. 

Carbol-broth 
Method. 

Polluted  waters  

+      -         ? 
•i-i      -21         t; 

+                  ? 

?8       3O          I 

Relatively  unpolluted  waters   

^6     38     2S 

37       6l       21 

The  English  Committee,  appointed  to  consider  the 
Standardization  of  Methods  for  the  Bacterioscopic  Exam- 
ination of  Water  (1904),  recommend  the  use  of  bile- 
salt  broth  or  glucose-formate  broth  for  preliminary 
enrichment,  and  suggest  that  the  incubation  be  carried 
out  anaerobically  at  42°  C. 

The  use  of  media  containing  bile  salts,  and  even  of 
undiluted  ox-bile  to  which  lactose  has  been  added  (Jack- 
son, 1906),  have  been  urged  by  various  American  bac- 
teriologists. With  sewages  and  heavily  polluted  waters 
the  lactose-bile  medium  has,  in  fact,  proved  superior  to 


Isolation  of  the  Colon  Bacillus.  93 

dextrose  broth.  When  a  large  proportion  of  sewage  is 
present  the  colon  bacilli  are  fresh  from  the  intestine  and 
apparently  able  to  resist  the  antiseptic  salts.  On  the  other 
hand,  the  large  numbers  of  other  bacteria  present  make 
the  danger  of  overgrowths  particularly  great.  It  is  possible, 
however,  that  direct  plating  on  litmus  lactose  agar  may 
prove  to  be  preferable,  even  to  the  bile  enrichment  method, 
for  waters  of  this  class.  With  waters  of  fair  quality,  such 
as  those  with  which  we  ordinarily  deal  in  sanitary  water 
analysis,  lactose  bile  is  open  to  the  same  objection  as 
phenol  broth,  though  in  less  degree.  It  inhibits  not  only 
the  overgrowing  forms  but  the  weaker  representatives  of 
the  B.  coli  group  itself;  and  the  net  effect  is  to  diminish 
positive  results.  In  an  examination  of  176  surface  waters 
in  eastern  Massachusetts,  using  lactose  bile  and  dextrose 
broth  in  parallel  for  preliminary  enrichment,  the  authors 
obtained  64  positive  results  by  the  former  method  against 
70  by  the  latter.  Longley  and  Baton  (1907),  from  their 
work  on  Potomac  water,  concluded  that  "the  value  of 
the  test  with  the  bile  lactose  on  unpolluted  or  slightly 
polluted  water,  such  as  we  have  to  deal  with  the  greater 
part  of  the  time,  is  uniformly  less  than  with  dextrose  broth, 
except  in  the  larger  quantities  of  water. " 

The  same  objection  applies  to  other  enrichment  methods 
which  involve  the  use  of  antiseptic  conditions  to  check 
the  development  of  overgrowing  bacteria.  Eijkman 
(1904)  suggested  incubation  at  46  degrees  as  furnishing  a 
more  rapid  differentiation  between  good  and  polluted 


94  Elements  of  Water  Bacteriology. 

waters  by  cutting  out  at  once  organisms  other  than  B. 
coli  which  fail  to  grow  at  this  high  temperature.  Chris- 
tian (1905),  Neumann  (1906),  and  Thomann  (1907) 
have  reported  good  results  by  the  use  of  this  method. 
It  remains,  however,  to  be  demonstrated  that  high  tem- 
peratures do  not  inhibit  colon  bacilli  in  slightly  polluted 
waters.  Nowack  (1907)  found  that  laboratory  cultures 
of  B.  coli  often  fail  to  produce  gas  in  Eijkman's  medium 
at  46  degrees,  unless  large  numbers  are  introduced.  With 
some  strains  an  inoculation  of  over  a  million  bacteria  was 
necessary  to  cause  gas  formation. 

It  appears  on  the  whole  that  the  safest  method  at  present 
available  is  the  dextrose  broth  enrichment  process  which 
alone  rests  on  the  sure  basis  of  a  great  number  of  observed 
coincidences  between  sanitary  inspection  and  bacteriolog- 
ical examination. 

When  it  is  desired  to  examine  samples  larger  than  i  c.c. 
for  B.  coli  it  becomes  necessary  to  modify  the  enrichment 
process  by  adding  the  nutrient  material  to  the  water 
instead  of  the  reverse.  For  this  purpose  phenol-dextrose 
broth  (consisting  of  broth  with  10  per  cent  dextrose,  5 
per  cent  peptone,  and  .25  per  cent  phenol)  may  be  added 
to  the  sample  of  water  to  be  enriched  as  suggested  by  Gage 
(Gage,  1901).  Generally  10  c.c.  of  the  broth  is  added  to 
100  c.c.  of  the  water.  The  sample  is  then  incubated  at 
37  degrees  for  twenty-four  hours,  and  if  at  the  end  of  that 
time  growth  has  taken  place,  a  cubic  centimeter  is  inocu- 
lated into  a  dextrose  tube.  If  this  tube  shows  gas 


Isolation  of  the  Colon  Bacillus.  95 

formation  after  twenty-four  hours  at  37  degrees,  a  litmus- 
lactose-agar  plate  is  made  and  the  other  diagnostic  tests 
applied. 

The  Committee  on  Standard  Methods  of  Water  Analysis 
(1905)  recommends  that  "for  ordinary  waters,  o.i,  i.o, 
and  10.0  c.c.  shall  be  used  for  the  colon  test.  For  sewage 
and  highly  polluted  surface-waters  smaller  quantities  shall 
be  used;  and  for  ground- waters,  filtered-waters,  etc.,  the 
quantities  shall  be  larger,  if  necessary,  to  obtain  positive 
results." 

Our  own  experience  has  been  that  it  is  not  specially 
advantageous  to  apply  the  colon  test  in  large  samples, 
since  the  significance  of  B.  coli  when  present  in  numbers 
less  than  i  per  c.c.  is  extremely  doubtful.  On  the  other 
hand,  the  danger  of  overgrowth  is  greatly  increased  in 
large  samples  and  negative  results  may  often  be  obtained 
when  the  organisms  are  really  present.  Hunnewell  and 
one  of  us  (Winslow  and  Hunnewell,  i9O2b)  found  that  of 
48  samples  of  certain  polluted  river  waters,  18  showed  B. 
coli  when  i  c.c.  was  inoculated  directly  into  dextrose  broth, 
while  in  only  4  cases  was  a  positive  result  obtained  after 
preliminary  treatment  of  100  c.c.  in  carbol  broth.  In  153 
samples  from  presumably  unpolluted  water,  B.  coli  was 
found  5  times  in  i  c.c.  and  n  times  by  the  examination  of 
the  larger  sample.  The  authors,  therefore,  concluded  as 
follows : 

"It  appears  evident  that  the  use  of  large  samples  in 
applying  the  colon  test  to  the  sanitary  analysis  of  drink- 


96  Elements  of  Water  Bacteriology. 

ing-water  is  not  advantageous.  In  comparing  the  results 
of  the  tests  in  i  c.c.  and  in  100  c.c.,  it  will  be  noted  that  the 
proportion  of  lactose  fermenting  organisms  and  of  colon 
bacilli  in  the  unpolluted  waters  was  more  than  doubled 
in  the  latter;  thus  waters  of  good  quality  are  more  likely 
to  be  condemned  by  the  use  of  large  samples.  On  the 
other  hand,  in  the  polluted  waters  a  considerable  propor- 
tion of  the  colon  bacilli  originally  present  were  lost  during 
the  incubation  of  the  large  samples,  so  that  waters  of 
bad  quality  actually  appeared  to  better  advantage  by  the 
use  of  100  c.c.  with  preliminary  incubation  in  phenol 
broth." 

Whipple  (Whipple,  1903)  notes  that  2.9  per  cent  of 
some  samples  of  water  examined  by  him  gave  positive 
tests  with  .1  c.c.  but  not  with  i  c.c.,  while  4.3  per  cent 
gave  positive  tests  with  .1  c.c.  or  i  c.c.  and  negative  tests 
with  10  c.c.  Again,  in  another  series  of  samples  exam- 
ined, of  those  which  gave  positive  tests  in  smaller  portions 
5.3  per  cent  were  negative  in  10  c.c.,  4.7  per  cent  in  100 
c.c.,  and  7.7  per  cent  in  500  c.c. 

In  our  ordinary  routine  at  the  Institute  we  use  one  cubic 
centimeter  sample  only,  inoculating  five  or  ten  tubes  in 
duplicate  with  that  amount.  In  this  way  we  ascertain 
whether  B.  coli  is  generally  or  rarely  present  in  one  cubic 
centimeter  of  the  suspected  water;  and  this  is  the  infor- 
mation of  greatest  practical  value.  If  absent  from  one 
cubic  centimeter  the  presence  of  the  organism  in  ten  cubic 
centimeters  would  not  lead  to  the  condemnation  of  the 


Isolation  of  the  Colon  Bacillus.  97 

water.  If  generally  present  in  one  cubic  centimeter  the 
water  may  be  considered  unsafe,  whether  the  colon  bacillus 
is  found  in  smaller  volumes  or  not.  For  special  studies 
of  self -purification,  etc.,  of  course  fractions  of  the  cubic 
centimeter  must  be  examined.  Litmus-lactose-agar  plates 
should  be  made  from  all  tubes  which  show  any  gas  what- 
ever. Fuller  and  Ferguson  (1905)  have  shown  that  B. 
coli  may  be  present  even  when  gas  formation  in  the 
enrichment  tube  is  quite  atypical.  Of  43  cultures  isolated 
by  these  observers  at  Indianapolis,  18  showed  less  than 
20  per  cent  of  gas  after  forty-eight  hours  in  the  enrich- 
ment tube,  ?nd  n  showed  less  than  10  per  cent. 

The  procedure  of  the  Committee  on  Standard  Methods 
of  Water  Analysis  (1905)  calls  for  a  forty-eight-hour  incu- 
bation of  the  preliminary  enrichment  tube.  Recent  expe- 
rience has,  however,  shown  that  a  twenty-four-hour  period 
gives  approximately  the  same  results  if  the  production  of 
gas  rather  than  any  specified  amount  of  gas  is  the  criterion 
of  a  positive  test.  Longley  and  Baton  (1907)  found  that 
of  1091  enrichment  tubes  giving  positive  tests  after  48 
hours  only  173  showed  no  gas  in  24  hours;  of  these  latter 
only  two  contained  B.  coli.  The  advantage  of  saving  a 
day  is  so  great  as  to  warrant  the  adoption  of  the  shorter 
period. 

In  all  practical  processes  of  examining  water  for  B.  coli 
one  essential  step  is  the  isolation  of  pure  cultures  upon 
the  litmus-lactose-agar  plate,  whether  the  plate  be  inocu- 
lated from  the  water  direct  or  from  a  preliminary  enrich- 


98  Elements  of  Water  Bacteriology. 

ment  culture.  In  the  first  case  a  measured  quantity  of 
water  is  added  and  the  number  of  colonies  of  B.  coli 
corresponds  to  the  number  of  bacteria  in  the  portion 
plated.  In  the  second  case,  since  the  enrichment  tube 
was  inoculated  with  a  known  amount  of  water  all  further 
work  is  purely  qualitative,  and  it  is  only  necessary  to 
obtain  such  a  number  of  colonies  upon  the  lactose  plate 
that  the  isolation  of  a  pure  culture  shall  be  easy.  In 
practice  the  following  procedure  has  been  found  generally 
successful:  After  the  dextrose  tubes  have  been  incubated 
for  twelve  to  twenty-four  hours  at  37  degrees,  from  those 
which  show  gas,  one  loopful  is  carried  over  to  a  tube  con- 
taining 10  c.c.  of  sterile  water,  and  of  this  water  one  loop- 
ful is  taken  for  the  inoculation  of  the  plate.  Ordinarily 
this  will  give  colonies  which  are  sufficiently  well  separated, 
but  a  second  plate,  inoculated  from  the  dilution  water 
with  a  straight  needle  instead  of  a  loop,  furnishes  a  de- 
sirable safeguard.  With  practice  it  is  possible  to  effect  a 
proper  seeding  more  rapidly  by  barely  touching  the  tip 
of  a  straight  needle  to  the  broth  in  the  fermentation  tube 
and  transferring  this  directly  to  the  agar.  The  touch 
must  be  a  very  light  one,  however,  or  the  colonies  on  the 
plate  will  be  too  thick  for  proper  isolation. 

The  litmus-lactose-agar  plates  made  in  this  manner 
should  be  incubated  for  from  twelve  to  twenty-four  hours 
at  the  body  temperature  (37  degrees),  at  the  end  of  which 
time,  if  B.  coli  is  present,  red  colonies  upon  a  blue  field 
will  be  visible.  The  litmus-lactose-agar  plate  may  be- 


Isolation  of  the  Colon  Bacillus.  99 

come  blue  again  after  forty-eight  hours',  owing  to  the  for- 
mation of  amines  and  ammonia  by  the  action  of  the 
bacteria  upon  the  nitrogenous  matter  present.  If  the 
dilution  is  too  low,  the  resulting  colonies  will  be  small 
and  imperfectly  developed,  making  it  difficult  to  be  sure 
of  pure  cultures  for  the  subsequent  tests.  A  great 
number  of  colonies  will  also  prevent  the  change  of  reac- 
tion from  acid  back  to  alkaline.  Since  many  bacteria 
ferment  lactose  with  the  formation  of  acid,  it  is  erroneous 
to  regard  all  colonies  as  those  of  B.  coli;  several  col- 
onies from  each  plate  should  be  isolated  upon  agar 
streaks  and  farther  studied  in  subculture. 

In  the  selection  of  those  red  colonies  which  are  to  be 
fished  from  the  litmus-lactose-agar  plate  the  appearance 
of  the  growths  must  be  closely  noted.  A  colony  of  irregu- 
lar contour,  surrounded  by  a  very  faint  area  of  reddening, 
will  probably  belong  to  some  member  of  the  B.  mycoides 
group  (Winslow  and  Nibecker,  1903);  small,  compact, 
bright-red  colonies  are  characteristic  of  the  streptococci, 
and  Gage  and  Phelps  (Gage  and  Phelps,  1903)  have 
pointed  out  that  of  these  there  are  two  types,  one  of  a 
brick-red  color,  and  of  such  consistency  as  to  be  readily 
picked  up  by  the  needle-point,  and  the  other  smaller  and 
of  an  intense  vermilion  color.  The  colonies  of  the  colon 
bacillus  are  usually  well  formed,  pulvinate  on  the  sur- 
face and  fusiform  when  growing  deeper  down. 

If  no  red  colonies  appear  on  the  litmus-lactose-agar 
plate  one  of  three  things  has  occurred.  There  may  be 


IOO  Elements  of  Water  Bacteriology. 

an  organism  present  which  forms  gas  in  dextrose  but  no 
acid  in  lactose;  there  may  be  present  forms  which 
individually  fail  to  attack  lactose  but  growing  together, 
symbiotically,  produce  gas  in  dextrose;  or  an  organism 
originally  present  and  capable  of  fermenting  both  sugars 
may  have  been  overgrown  and  lost  in  the  enrichment 
tube.  If  plates  are  made  on  the  first  appearance  of  gas 
the  likelihood  of  the  latter  possibility  will  be  reduced  to 
a  minimum.  Neither  of  the  first  two  contingencies  has 
any  sanitary  significance;  as  we  shall  see  later,  bacteria 
which  ferment  detrose  and  not  lactose  are  not  specially 
characteristic  of  pollution.  In  any  case,  therefore,  the 
absence  of  red  colonies  on  the  agar  plate  may  be  con- 
sidered a  negative  result.  If  red  colonies  are'  present 
they  must  be  subcultured  and  examined  further. 

The  agar  streak  made  from  the  litmus-lactose-agar  plate 
shows  after  twenty-four  hours  certain  marked  character- 
istics. The  most  distinct  types  are  two,  the  abundant, 
first  translucent,  later  whitish  and  cheesy  growth,  cover- 
ing nearly  the  whole  surface  of  the  agar,  characteristic  of 
B.  coli  and  its  allies,  and  a  very  faint  growth,  either  con- 
fined strictly  to  the  streak  or  made  up  of  faint  isolated 
colonies,  dotted  here  and  there  over  the  surface.  The 
latter  cultures  are  typical  of  the  sewage  streptococci,  and 
a  microscopic  examination  will  generally  settle  their  status 
at  once.  Of  the  more  luxuriant  growths,  some  of  which 
are  stringy  to  the  needle,  many  will  generally  prove  to  be 
atypical,  and  if  any  of  the  weakly  fermenting  forms  (B. 


Isolation  of  the  Colon  Bacillus.  101 

mycoides)  are  present,  a  dull  wrinkled  growth  will  be 
produced. 

Having  submitted  the  sample  of  suspected  water  to  a 
preliminary  enrichment  process,  and  having  isolated  pure 
cultures  of  suspicious  organisms  from  the  litmus-lactose- 
agar  plate,  the  third  step  is  the  examination  of  the  specific 
reactions  of  the  organisms  thus  obtained.  Just  what 
characters  to  use  in  denning  the  "colon  bacillus"  is  a 
matter  of  prime  importance.  The  whole  question  of 
species  among  the  bacteria  is  an  extremely  complex  one, 
since  around  each  definite  species  are  grouped  forms 
differing  from  the  type  in  one  or  two  of  its  characteristics. 

As  Whipple  says  (Whipple,  1903),  "The  type  form  of 
Bacillus  coli  is  one  which  can  be  defined  within  reason- 
ably narrow  limits,  but  when  the  organism  has  been  away 
from  its  natural  habitat  for  varying  periods  of  time,  and 
has  existed  under  abnormal  conditions,  its  ability  to  react 
normally  to  the  usual  tests  appears  to  be  greatly  impaired. 
Its  power  to  reduce  nitrates  may  be  lost,  or  on  the  other 
hand  may  be  increased;  its  power  to  produce  indol  may 
be  lost,  or  on  the  other  hand  it  may  be  increased;  its 
power  to  coagulate  milk,  even,  is  sometimes  reduced, 
although  seldom  entirely  lost;  its  power  to  ferment  car- 
bohydrates may  be  altered  so  that  the  amount  of  gas 
obtained  in  a  fermentation  tube,  as  well  as  its  ratio  of 
H  to  CO 2,  is  quite  abnormal.  But  in  spite  of  all  these 
facts,  the  bacillus  tested  may  have  been  originally  a  true 
Bacillus  coli." 


IO2  Elements  of  Water  Bacteriology. 

It  is,  of  course,  not  always  certain  that  organisms  resem- 
bling B.  coli,  but  failing,  for  example,  to  reduce  nitrates  or 
to  form  indol,  have  been  derived  from  typical  colon  bacilli 
by  any  recent  process  of  modification.  Organisms  of 
this  sort  may  be  found  which  for  generations  breed  true 
to  their  characteristics  and  are  apparently  definitely  dif- 
ferent in  one  property  or  another  from  the  true  B.  coli. 

The  more  of  such  atypical  forms  which  are  included 
the  greater  will  be  the  number  of  positive  isolations.  The 
definition  of  this  or  any  other  bacterial  species  is  more 
or  less  arbitrary;  we  consider  as  true  colon  bacilli  those 
which  fulfill  a  particular  set  of  tests,  and  class  as  pseudo- 
colon  organisms  those  which  do  not.  If  we  find,  having 
established  such  an  arbitrary  standard,  that  the  colon 
bacillus,  as  determined  by  it,  is  found  in  waters  known  to 
be  polluted,  and  not,  as  a  rule,  in  those  known  to  be  free 
from  pollution,  the  sanitarian  can  afford  to  ignore  the 
theoretical  question  of  specific  values  and  make  confident 
use  of  the  practical  test.  In  order  that  results  may  rest 
on  a  sound  basis  of  comparable  data  for  various  waters, 
it  is  of  course  however  essential  that  a  standard  set  of 
reactions  should  be  agreed  upon  by  sanitary  bacteriologists. 

After  a  considerable  period  of  uncertainty,  in  which  each 
observer  used  the  procedure  which  happened  to  appeal  to 
him,  the  attainment  of  comparative  results  has  been  made 
possible  by  the  establishment  of  standard  methods  of 
procedure  by  bodies  of  authoritative  position,  both  in 
England  and  America.  In  1904  an  English  Committee, 


Isolation  of  the  Colon  Bacillus.  103 

appointed  to  consider  the  Standardization  of  Methods 
for  the  Bacterioscopic  Examination  of  Water,  presented 
a  series  of  obligatory  tests  and  optional  tests;  and  in  1905 
the  Committee  on  Standard  Methods  of  Water  Analysis 
of  the  American  Public  Health  Association  drew  up  a  set 
of  diagnostic  characters  for  B.  coli.  The  latter  corresponds 
in  general  with  the  plan  developed  by  the  Massachusetts 
State  Board  of  Health  (Massachusetts  State  Board  of 
Health,  1899)  and  long  in  use  at  the  Massachusetts 
Institute  of  Technology. 

It  involves  the  use  of  the  following  seven  tests : 

DIAGNOSTIC  CHARACTERS  FOR  B.  COLI. 

1.  Typical    morphology  —  non-sporing    bacillus,    rela- 
tively small  and  often  quite  thick. 

2.  Motility  —  when  a  young  broth  or  gelatin  culture  is 
examined. 

3.  Fermentation  of  dextrose  broth, with  the  formation  of 
about  50  per  cent  of  gas,  of  which  about  one-third  (CO2) 
is  absorbed  by  a  two  per  cent  solution  of  sodium  hydrate. 

4.  Coagulation  of  milk,  with  the  production  of  acid,  in 
48  hours  or  more  at  37°  C.,  either  spontaneously  or  upon 
boiling. 

5.  Non-liquefaction  of  gelatin. 

6.  Production  of  indol  in  peptone  solution. 

7.  Reduction  of  nitrates. 

The    English    standard    procedure    corresponds    quite 
closely   to   this    (Committee   appointed   to   consider   the 


IO4  Elements  of  Water  Bacteriology. 

Standardization  of  Methods  for  the  Bacterioscopic  Exam- 
ination of  Water,  1904),  although  it  differs  from  the 
American  method  in  certain  respects. 

The  American  Public  Health  Association  standard 
procedure,  as  defined  above,  has,  in  the  main,  proved 
satisfactory.  In  two  respects,  however,  it  needs  modi- 
fication. 

In  the  first  place,  the  requirement  that  motility  should  be 
demonstrated  is  a  burdensome  and  needless  one.  Motility 
is  a  fluctuating  and  uncertain  property  and  one  which 
frequently  requires  repeated  preliminary  cultivations  to 
make  it  manifest.  Furthermore,  non-motile  colon  bacilli 
are  common  in  the  intestine  and  are  probably  as  char- 
acteristic of  intestinal  pollution  as  the  motile  forms. 
McWeeney  (1904)  found  non-motile  B.  coli  abundant 
in  feces  and  observed  cases  where  the  organisms  were 
motile  at  20  degrees  and  not  at  37  degrees.  He  quotes 
Stocklin  as  having  found  116  non-motile  strains  among 
300  otherwise  normal  B.  coli  from  feces.  Evidence  that 
non-motile  bacteria,  otherwise  resembling  B.  coli,  occur 
in  unpolluted  water  would  furnish  the  only  basis  for 
requiring  this  test  as  a  routine  procedure.  No  such 
evidence  exists.  The  great  body  of  data  which  connects 
the  presence  of  B.  coli  with  pollution  includes  all  B.  coli 
whether  motile  or  not,  since  scarcely  any  bacteriologists 
observe  this  property  in  actual  practice. 

Another  point  in  the  Diagnostic  Tests  of  the  Committee 
on  Standard  Methods  which  requires  modification  in  the 


Isolation  of  the  Colon  Bacillus.  105 

light  of  more  recent  knowledge  is  the  requirement  that 
dextrose  broth  shall  be  fermented  "with  the  formation  of 
about  50  per  cent  of  gas,  of  which  about  one-third  (CO2) 
is  absorbed  by  a  two  per  cent  solution  of  sodium  hydrate." 
Stamm  (1906)  and  others  have  pointed  out  that  the  ratio 
of  carbon  dioxide  to  hydrogen  changes  with  the  age  of  the 
culture.  At  first  the  proportion  of  the  former  to  the  latter 
is  as  two  to  one,  and  later,  in  the  same  tube,  the  ratio  is 
reversed.  More  recently,  Longley  and  Baton  (1907),  in 
one  of  the  ablest  and  most  fruitful  of  recent  contributions 
to  water  bacteriology,  have  made  it  clear  that  neither  of 
these  quantitative  determinations  is  of  importance.  They 
show,  first,  that  the  total  amount  of  gas  formed  by  B.  coli 
varies  w  idely,  from  10  to  80  per  cent,  the  mode  of  the  curve 
being  found  not  at  50  but  at  35  per  cent.  Secondly,  they 
show  that  the  proportion  of  carbon  dioxide  present  is  a 
function  of  the  total  amount  of  gas.  They  find  that  when 
grown  in  an  atmosphere  of  CC>2,  B.  coli  produces  a  gas 
which  consists  of  about  3  parts  of  carbon  dioxide  to  one 
of  hydrogen.  Assuming  that  the  gas  originally  formed  by 
B.  coli  has  always  about  this  composition,  and  that  the 
absorption  of  CC>2  by  the  medium  is  the  chief  cause  of  the 
differences  observed  in  the  gas  which  collects  in  the  closed 
arm,  the  gas  ratio  would  vary  directly  with  the  amount 
of  total  gas;  the  more  rapidly  gas  is  formed,  the  greater 
the  proportion  of  CO2  remaining  unabsorbed.  Calcula- 
tion on  this  basis  gives  a  curve  very  close  to  the  observed 
data,  and  the  conclusion  of  Longley  and  Baton  that  the 


io6  Elements  of  Water  Bacteriology. 

gas  ratio  is  a  function  of  the  total  quantity  of  gas  seems 
us  justified.  The  determination  of  the  gas  ratio  may 
therefore  be  omitted,  and  any  tests  which  show  from  10  to 
80  per  cent  gas  in  dextrose  broth  in  forty-eight  hours  may 
be  considered  positive. 

For  recording  the  results  of  the  various  tests  applied 
in  sanitary  water  examination,  the  appended  blank  form 
has  been  in  use  at  the  Massachusetts  Institute  of  Tech- 
nology. On  the  upper  part  of  the  sheet  are  noted  the 
results  of  the  gelatin  count  and  the  litmus-agar  count  at 
37  degrees.  In  the  second  column  the  number  of  acid- 
formers  is  placed  in  brackets  after  the  total  numbers. 
The  lower  part  is  used  for  the  B.  coli  isolation. 

MASSACHUSETTS   INSTITUTE   OF  TECHNOLOGY. 
Bacteriological  Examination  of  Water. 

Sample  No.  Date  of  collection 

Examined  by  Hour  of  collection 

Place  of  collection  Remarks 

Number  of  Bacteria. 

Gelatin-plate  cultures.          No.  colonies         Agar-plate  cultures  at  37°. 
48  hours.  per  c.c.  No.  colonies  per  c.c. 

24  hours 

Average  Average 

Remarks  Remarks 


B.  Coli  Isolation. 


Preliminary  fermentation  tube 
Litmus-lactose-agar  plates 
Agar  streak  culture 
Fermentation  tube 
Nitrate  test 
Milk  test 
Indol  reaction 
Gelatin-stab  culture 
Remarks 


Isolation  of  the  Colon  Bacillus.  107 

Opposite  the  words  "  Preliminary  fermentation  tube  " 
are  recorded  by  plus  or  minus  signs  the  results  of  the  first 
enrichment  test  in  the  five  or  ten  tubes  inoculated.  Under 
the  columns  headed  by  plus  signs  the  presence  or  absence 
of  typical  red  colonies  on  the  litmus-lactose-agar  plate  is 
similarly  indicated.  The  third  line  serves  for  the  results 
of  the  microscopic  and  macroscopic  appearance  of  the 
agar  streak.  If  this  gives  the  proper  type  of  growth  and 
rod-shaped  organisms  are  found,  a  dextrose  fermentation 
tube,  a  nitrate  tube,  -a  milk  tube,  a  peptone  tube,  and  a 
gelatin  stab  are  inoculated  from  it. 

After  forty-eight  hours  incubation  at  37  degrees,  the 
dextrose  broth  and  milk  tubes  are  examined  and  the 
results  recorded  in  the  proper  columns  by  plus  and  minus 
signs.  The  amount  of  gas  in  the  closed  arm  of  the  dex- 
trose tube  may  be  conveniently  measured  by  the  Frost 
gasometer  (Frost,  1901).  If  a  measurement  of  the  gas 
ratio  is  desired  a  few  centimeters  of  strong  sodium  or 
potassium  hydrate  are  added  and  mixed  with  the  broth 
by  cautiously  tipping  the  tube;  a  second  measurement 
determines  the  amount  of  gas  absorbed  (assumed  to  be 
CO2).  The  gas  should  first  fill  from  one-third  to  two- 
thirds  of  the  closed  arm,  and  about  one-third  of  the  total 
amount  should  be  absorbed. 

The  milk  tube  is  tested  by  heating  to  boiling  over  the 
free  flame;  if  coagulation  occurs  the  test  is  considered 
positive. 

After  ninety-six  hours  incubation  at  37   degrees  the 


io8  Elements  of  Water  Bacteriology. 

peptone  solution  is  examined  for  indol  by  adding  i  c.c.  of  a 
.02  per  cent  solution  of  sodium  or  potassium  nitrite  and 
i  c.c.  of  a  i  to  i  solution  of  sulphuric  acid.  Both  the  tube 
and  the  reagents  should  be  cooled  on  ice  before  mixing, 
and  the  tube  should  be  left  in  a  cool  place  for  an  hour 
afterward  to  allow  time  for  the  characteristic  rose-red 
color  of  nitroso -indol  to  develop.  At  the  same  time 
(after  four  days)  the  nitrate  tube  is  tested  for  nitrites  by 
adding  a  drop  of  each  of  the  following  solutions  in  suc- 
cession: 

A.  Sulphanilic  acid .5  gram 

Acetic  acid  (25%  sol.) 150-0  c.c. 

B.  Naphthylamine  chloride .1  gram 

Distilled  water 20.00  c.c. 

Acetic  acid  (25%  sol.) .-   .    .  150.0    c.c. 

A  red  or  violet  coloration  indicates  the  presence  of  nitrites. 

In  making  the  nitrite  and  indol  tests  it  is  important 
to  remember  the  possibility  that  appreciable  amounts  of 
nitrite  may  be  present  in  the  media  —  either  derived  from 
the  air  or  from  the  use  of  impure  peptone  in  the  indol 
solution  (Wherry,  1905).  In  the  case  of  the  nitrite  reac- 
tion, control  tubes  should  always  be  tested  from  the  same 
batch  of  media  and  only  a  distinct  red  color  should  be 
considered  positive. 

The  gelatin  tube  should  be  kept  at  20  degrees  for  ten 
days.  It  seems  undesirable  in  practice  to  prolong  the 
test  much  beyond  this  point,  although  some  slowly  lique- 
fying organisms  are  doubtless  included,  which  would  be 
thrown  out  by  a  longer  incubation.  The  extent  of  this 


Isolation  of  the  Colon  Bacillus. 


109 


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no  Elements  of  Water  Bacteriology. 

source  of  error  as  well  as  the  relative  importance  of  the 
various  other  diagnostic  tests  is  well  shown  in  the  table 
(page  109)  of  the  results  obtained  at  Lawrence  during  a 
period  of  eighteen  months. 

It  should  be  noted  that  considerable  differences  often 
appear  in  these  biochemical  reactions  between  tubes  of 
the  same  batch  of  a  medium,  inoculated  with  approxi- 
mately the  same  amount  of  the  same  culture.  This  has 
been  shown  very  markedly  for  the  amount  of  the  gas  and 
the  proportion  of  carbon  dioxide  in  the  dextrose  tube  by 
Fuller  and  Johnson  (Fuller  and  Johnson,  1899),  Penning- 
ton  and  Kiisel  (Pennington  and  Kiisel,  1900),  Gage 
(Gage,  1902),  and  one  of  ourselves  (Winslow,  1903). 
Variations  in  nitrate  reduction  are  often  even  more 
marked,  one  tube,  perhaps,  showing  a  strong  reaction 
and  another  none.  In  important  cases,  therefore,  it  is 
desirable  to  inoculate  the  subcultures  in  duplicate. 

These  anomalies  are  most  frequent  with  cultures  freshly 
isolated  from  water,  and  they  may  often  be  avoided,  as 
Fuller  and  Johnson  (1899)  have  shown,  by  subjecting  the 
organism  to  a  process  of  preliminary  cultivation.  For 
this  purpose  the  American  Public  Health  Association 
Committee  recommends  three  successive  cultivations  in 
broth  at  20  degrees,  each  of  24  hours  duration,  inoculation 
from  the  last  broth  tube  of  a  gelatine  plate  which  is  incu- 
bated for  48  hours  at  20  degrees,  inoculation  of  an  agar 
streak  from  one  colony  on  the  plate  and  incubation  of 
this  streak  for  48  hours  at  20  degrees. 


Isolation  of  the  Colon  Bacillus.  1 1 1 

In  general  routine  work  there  will  scarcely  be  time  to 
carry  out  preliminary  enrichment  processes  of  this  sort, 
and  cultures  which  fail  to  reduce  nitrates  or  to  form  indol, 
or  which  do  not  form  sufficient  acid  in  milk  to  coagulate 
it,  must  be  classed  as  atypical  colon  bacilli  or  "  Paracolon  " 
bacilli.  The  interpretation  of  results  of  this  kind  will  be 
discussed  in  Chapter  IX.  It  may  be  noted  in  passing  that 
the  results  of  the  test  for  B.  coli  will  fall  under  four  general 
heads:  If  the  preliminary  dextrose  tube  or  the  litmus- 
lactose-agar  plate  fail  to  show  fermentation,  the  test  is 
negative.  If  these  are  positive  and  gelatin  is  liquefied, 
we  are  dealing  with  a  member  of  the  B.  cloacae  group. 
If,  on  the  other  hand,  gelatin  is  not  liquefied,  the  organ- 
ism is  a  typical  B.  coli  if  it  meets  the  standard  require- 
ments of  the  nitrate,  indol,  and  milk  tests.  If  not  it 
must  be  classed  as  an  allied  "  atypical  "  form. 


CHAPTER   VII. 

SIGNIFICANCE   OF  THE   PRESENCE   OF    B.    COLI   IN   WATER. 

FIFTEEN  years  ago  the  B.  coli  of  Escherich  occupied  a 
position  of  very  great  prominence  in  the  eyes  of  sani- 
tarians. If  it  was  not  considered  to  be  in  itself  a  danger- 
ously pathogenic  germ,  it  was  at  least  regarded  as  a 
suspiciously  close  relation  of  the  typhoid  organism.  At 
this  time,  the  alleged  presence  of  either  of  these  forms 
was  quite  sufficient  to  condemn  a  water-supply. 

Investigation  soon  showed,  however,  that  the  Bacillus 
coli  was  by  no  means  confined  to  the  human  intestine. 
Dyar  and  Keith  (Dyar  and  Keith,  1893)  found  it  to  be 
the  prevailing  intestinal  form  in  the  cat,  dog,  hog,  and 
cow.  About  the  same  time,  Fremlin  (Fremlin,  1893) 
found  colon  bacilli  in  the  feces  of  dogs,  mice,  and  rabbits, 
but  not  in  those  of  rats,  guinea  pigs,  and  pigeons.  Smith 
(Smith,  1895)  recorded  the  presence  of  the  organism,  in 
almost  pure  cultures,  in  the  intestines  of  dogs,  cats,  swine, 
and  cattle;  and  he  also  found  it  in  the  organs  of  fowls 
and  turkeys  after  death.  Brotzu  (Brotzu,  1895)  reported 
B.  coli  and  allied  forms  as  very  abundant  in  the  intestine 
of  the  dog;  and  Belitzer  (Belitzer,  1899)  isolated  typical 
colon  bacilli  from  the  intestinal  contents  of  horses,  cattle, 


The  Significance  of  B.  Coli  in    Water.         113 

swine,  and  goats.  Moore  and  Wright  (Moore  and  Wright, 
1900)  recorded  the  finding  of  the  colon  bacillus  in  the 
horse,  cow,  dog,  sheep,  and  hen,  and  in  a  later  report 
(Moore  and  Wright,  1902)  they  noted  its  occurrence  in 
swine  and  in  some  but  not  all  the  specimens  of  rabbits 
examined.  In  frogs  it  was  not  found.  Eyre  (1904) 
has  more  recently  isolated  typical  B.  coli  from  the  intes- 
tines of  mice,  rats,  guinea  pigs,  rabbits,  cats,  dogs,  sheep, 
goats,  horses,  cows,  hens,  ducks,  pigeons,  sparrows, 
divers,  gulls,  and  fish  of  various  sorts.  Houston  (1904) 
found  B.  coli  abundant  in  the  feces  of  gulls,  as  might  be 
expected  from  their  feeding  habits.  Houston  (1905) 
and  other  recent  observers  have  found  it  impossible, 
even  by  the  use  of  elaborate  series  of  fermentation  tests, 
to  distinguish  human  B.  coli  from  those  found  in  animals. 
Savage  (1906)  compared  colon-like  organisms  isolated 
from  the  intestines  of  swine,  cattle,  horses,  and  sheep 
with  those  of  human  origin  in  respect  to  their  action  upon 
lactose,  dulcite,  mannite,  raffinose,  glycerine,  maltose, 
galactose,  laevulose,  saccharose,  starch  and  cellulose; 
but  he  failed  to  find  any  general  correlations  between 
habitat  and  biochemical  powers. 

In  cold-blooded  animals  the  occurrence  of  B.  coli  is 
less  constant.  Negative  results  in  the  frog  and  positive 
results  in  certain  fishes  have  just  been  quoted.  Amyot 
(1902)  failed  to  find  the  organism  in  the  intestines  of 
23  fish  representing  14  species.  Johnson,  on  the  other 
hand  (Johnson,  1904),  in  the  examination  of  the  stomach 


114  Elements  of  Water  Bacteriology. 

and  intestines  of  67  fish  caught  in  the  polluted  Illinois 
and  Mississippi  Rivers,  isolated  B.  coli  47  times.  He, 
concluded  from  these  results  that  the  migration  of  fish 
from  a  contaminated  stream  or  lake  to  an  unpolluted  one 
may  explain  the  occasional  finding  of  B.  coli  in  small 
samples,  or  the  more  regular  detection  of  it  in  large 
volumes  of  the  water. 

Many  bacteriologists  have  gone  further  and  affirmed 
that  the  colon  bacillus  was  not  a  form  characteristic  of 
the  intestine  at  all,  but  a  saprophyte  having  a  wide  distri- 
bution in  nature.  The  first  of  this  school,  perhaps, 
was  Kruse  (Kruse,  1894),  who  in  1894  protested  against 
the  arbitrary  conclusions  drawn  from  the  colon  test  as 
then  applied.  He  pointed  out  that  the  characters  usually 
observed  marked  not  a  single  species  but  a  large  group 
of  organisms.  As  ordinarily  defined,  he  added,  "the 
Bacterium  coli  is  in  no  way  characteristic  of  the  feces  of 
men  or  animals.  Such  bacteria  occur  everywhere,  in 
air,  in  earth,  and  in  the  water,  from  the  most  different 
sources."  Even  if  the  relations  to  milk  and  sugar  media 
be  considered,  "  micro-organisms  with  these  characteristics 
are  also  widespread."  Dr.  Kruse  gave  no  experimental 
data  on  which  his  opinion  was  based.  In  the  same  year 
Beckmann  (Beckmann,  1894)  isolated  a  bacilhis  which 
he  identified  by  pretty  thorough  tests  as  B.  coli  from  the 
city  water  of  Strassburg,  a  ground-water  which  he  believed 
could  by  no  possibility  be  subject  to  fecal  contamination. 
Large  quantities  of  water  were  used  for  the  isolation. 


The  Significance  of  B.  Coli  in    Water.          115 

Refik  (Refik,  1896)  recorded  the  constant  presence  of 
colon  bacilli  in  water  of  all  sorts,  public  supplies,  wells, 
cisterns,  and  springs  in  the  neighborhood  of  Constanti- 
nople, but  the  only  characters  which  these  "colon  bacilli" 
exhibited  in  common  were  the  " classical  growth"  upon 
potato,  the  possession  of  less  than  8  cilia,  and  the  power 
of  active  development  on  certain  media  upon  which  the 
typhoid  bacillus  did  not  grow.  A  more  careful  and  sig- 
nificant piece  of  work  on  the  same  line  was  published  by 
Poujol  in  the  succeeding  year.  This  author  reported 
(Poujol,  1897)  *ne  isolation  of  B.  coli  from  22  out  of  34 
waters  studied  by  him  in  relation  to  their  use  as  public 
supplies.  The  waters  were  from  various  sources  — 
springs,  wells,  and  rivers  —  but  all  were  of  fair  quality 
and  many  quite  free  from  any  possibility  of  contamina- 
tion. Samples  of  100  c.c.  were  used  for  analysis;  in  the 
only  case  in  which  a  smaller  amount  was  also  tested, 
broth  inoculated  with  10  drops  of  the  water  and  placed 
at  45°  C.  remained  sterile.  The  author  concluded 
that  "fecal  contamination  can  only  exceptionally  be 
invoked  to  explain  the  presence  of  B.  coli  in  water.  As 
the  bacteria  of  the  subterranean  water  are  contributed 
to  it  from  the  surface  of  the  earth  by  the  water  which 
filters  downward,  I  am  rather  inclined  to  believe  in  a 
general  diffusion  of  B.  coli  either  on  the  surface  of  the 
earth,  where  it  might  be  deposited  with  the  dust  of  the 
air,  or  in  the  superficial  layers  of  the  earth,  which  may 
form  one  of  its  normal  habitats."  Therefore,  the  author 


n6  Elements  of  Water  Bacteriology. 

considered  that  caution  should  be  exercised  in  condemn- 
ing a  water  on  account  of  the  presence  of  B.  coli,  except, 
as  he  added,  "for  those  cases  where  it  exists  in  consider- 
able quantity." 

Certain  Italian  observers  appear  to  have  come  to  even 
less  conservative  conclusions.  Abba  (Abba,  1895)  found 
colon  bacilli  constantly  present  in  unpolluted  waters  near 
Turin.  Moroni  (Moroni,  1898;  Moroni,  1899)  reported 
the  examination  of  numerous  deep  and  shallow  wells  and 
unpolluted  springs  about  Parma,  as  well  as  of  the  public 
water-supply  of  the  city,  and  concluded  that  the  colon 
bacillus  was  a  water  form  and  had  no  sanitary  significance. 
The  characters  used  for  the  identification  of  the  species 
in  this  case  were  fairly  exhaustive,  but  both  Abba  and 
Moroni  used  liter  samples  for  analysis. 

Levy  and  Bruns  (Levy  and  Bruns,  1899)  gave  a  new 
turn  to  the  discussion  by  emphasizing  the  importance  of 
animal  inoculation,  already  suggested  by  Blachstein 
(Blachstein,  1893)  and  others.  They  claimed  that  the 
existence  of  numerous  para-colon  and  par^-typhoid  or- 
ganisms in  air,  in  dust,  and  in  unpolluted  water  made  it 
impossible  to  decide  by  ordinary  bacteriological  methods 
whether  true  colon  bacilli  were  present  in  water  or  not. 
In  no  case,  however,  did  representatives  of  the  colon  group 
isolated  by  them  from  water  kill  a  guinea  pig,  even  when 
i  or  2  c.c.  were  injected  intraperitoneally.  The  authors, 
therefore,  considered  pathogenicity  as  an  attribute  belong- 
ing only  to  the  true  B.  coli  of  the  intestine.  This  paper 


The  Significance  of  B.  Coli  in    Water.         117 

aroused  Professor  Kruse's  pupil,  Weissenfeld,  to  a  pub- 
lication, in  which  the  position  of  the  Bonn  school  was 
carried  to  an  extreme.  Weissenfeld  reported  (Weissenfeld, 
1900)  the  analysis  of  30  samples  of  water  supposedly  pure, 
and  of  26  samples  considered  to  be  contaminated.  In 
each  case  a  single  centimeter  sample  was  first  incubated 
in  Parietti  broth,  and  if  no  growth  occurred,  larger  samples 
of  half  a  liter  or  a  liter  were  examined.  Colon  bacilli 
were  found  in  all  the  samples,  and  the  pathogenicity 
varied  independently  of  the  source  of  the  water.  The 
author  concluded  that  "  the  so-called  Bacterium  coli 
may  be  found  in  waters  from  any  source,  good  or  bad, 
if  only  a  sufficiently  large  quantity  of  the  water  be  taken 
for  analysis." 

With  regard  to  the  question  of  pathogenicity  as  a 
diagnostic  test  for  intestinal  B.  coli,  there  is  little  doubt 
of  the  correctness  of  Weissenfeld's  conclusions.  This 
property  is  so  variable  as  to  have  no  important  value. 
Colon  bacilli  freshly  isolated  from  the  intestine  are  fre- 
quently non-virulent,  and  Savage  (1903*)  and  others 
have  shown  that  there  is  in  general  no  correlation  between 
pathogenic  power  and  direct  or  indirect  intestinal  origin. 
On  the  other  hand  Weissenfeld's  work  entirely  fails  to 
show  that  the  colon  bacillus,  pathogenic  or  non-patho- 
genic, is  a  normal  inhabitant  of  unpolluted  waters.  In 
the  first  place  it  should  be  noted  that  the  characters  used 
by  this  investigator  for  defining  the  "  so-called  Bacterium 
coli "  were  absolutely  inadequate.  He  classed  under 


1 1 8  Elements  of  Water  Bacteriology. 

that  head  all  bacilli  of  medium  size,  which  formed  grape- 
vine-leaf colonies  on  gelatin  and  gas  in  sugar  agar,  which 
were  more  or  less  motile,  or  rarely  non-motile,  and  which 
were  decolorized  by  the  Gram  method.  As  regards 
coagulation  of  milk  and  formation  of  indol,  "  the  bacteria 
isolated  differed."  In  the  second  place  it  is  difficult  to  see 
how  the  author  could  possibly  have  believed  that  his 
experiments  proved  the  isolation  of  the  colon  bacillus  to  be 
"  useless  as  an  aid  in  the  sanitary  examination-of  water," 
as  the  title  of  the  paper  runs.  Even  his  own  work  fur- 
nishes strong  evidence  to  the  contrary.  In  24  of  the  26 
samples  from  bad  sources,  he  isolated  his  imperfectly 
denned  colon  bacilli  from  i  c.c.  of  the  water,  while  in 
only  8  of  the  30  samples  of  good  waters  could  he  find  such 
organisms  in  that  quantity. 

The  work  of  certain  recent  observers  has  suggested 
the  possibility  that  the  colon  bacillus  may  live  in  a  semi- 
parasitic  fashion  on  plants  as  well  as  on  animals.  Of 
a  series  of  47  cultures  of  lactic-acid  bacteria,  recently 
examined  by  one  of  ourselves  (Prescott,  1902*;  Prescott, 
1903;  Prescott,  1906),  25  were  found  to  give  the  reactions 
of  B.  coli.  These  organisms  were  isolated  chiefly  from 
cereals  and  products  of  milling,  such  as  flour,  bran,  corn- 
meal,  oats,  barley,  etc.,  while  others  ^were  in  technical  use 
for  producing  the  lactic  fermentation.  There  is  no  evi- 
dence that  any  of  these  organisms  were  of  intestinal  origin, 
and  yet  they  possessed  all  the  characters  of  typical  colon 
bacilli,  even  to  the  pathogenic  action  when  inoculated 


The  Significance  of  B.  Coli  in    Water.         119 

into  guinea  pigs.  In  Germany,  Papasotiriu  (Papasotiriu, 
1901)  was  meanwhile  carrying  on  almost  exactly  similar 
investigations  to  Prescott's,  with  identical  results. 

Other  testimony  is  somewhat  conflicting  with  regard 
to  the  occurrence  of  B.  coli  on  plants.  Klein  and  Houston 
(1900)  reported  the  finding  of  typical  colon  bacilli  in  only 
3  out  of  24  samples  of  wheat  and  oats  obtained  from  a 
wholesale  house;  rice,  flour,  and  oatmeal  bought  at  two 
different  retail  shops  gave  B.  coli  in  all  three  cereals 
in  one  case  and  none  in  the  other.  Clark  and  Gage 
(1903)  were  unable  to  isolate  B.  coli  from  standing  grains. 
Gordan  (1904)  could  not  find  B.  coli  in  .1  and  .01  mg. 
samples  of  clean  bran,  but  isolated  it  easily  from  that 
of  poor  quality.  Winslow  and  Walker  (1907)  have 
recently  reported  the  examination  of  178  samples  of  grain 
and  40  samples  of  grasses  for  B.  coli  without  success.  On 
the  other  hand,  Diiggeli  (1904)  found  B.  coli  among 
the  bacteria  occurring  on  the  leaves  of  growing  plants, 
although  it  was  not  one  of  the  most  abundant  species. 
Barthel,  too  (Barthel,  1906),  found  B.  coli  widely  dis- 
tributed on  plants  from  both  cultivated  and  uncultivated 
regions. 

These  results  raise  the  interesting  questions:  Is  it 
possible  that  the  lactic-acid  bacilli  and  the  similar  forms 
found  on  plants  have  been  indirectly  derived  from  animal 
intestines,  having  "escaped  from  cultivation,"  as  the 
botanists  say?  Or  is  the  converse  true,  namely,  that  all 
colon  bacilli  are  simply  plant  parasites  which  have  found 


I2O  Elements  of  Water  Bacteriology \ 

in  the  warm  intestinal  canal,  richly  supplied  with  food,  a 
favorable  habitat? 

The  answer  to  these  questions  is  of  much  theoretical 
interest,  but  need  not  be  further  considered  here.  The 
practical  sanitary  conclusions  to  be  drawn  are  as  follows: 

1.  Bacteria  corresponding  in  every  way  to  B.  coli  are 
by  no  means  confined  to  animal  intestines,  but  are  widely 
distributed  elsewhere  in  nature. 

2.  The  finding  of  a  few  colon  bacilli  in  large  samples 
of  water,   or  its   occasional  discovery  in  small  samples, 
does  not  necessarily  have  any  special  significance. 

3.  The  detection  of  B.  coli  in  a  large  proportion  of 
small  samples   (i  c.c.  or  less)  examined  is  imperatively 
required  as  an  indication  of  recent  sewage  pollution. 

4.  The  number  of  colon  bacilli  in  water  rather  than 
their  presence  should  be  used  as  a  criterion  of  recent 
sewage  pollution. 

With  these  qualifications  the  value  of  the  colon  test 
was  never  more  firmly  established  than  it  is  to-day. 
Whether  or  not  originally  a  domesticated  form,  it  is  clear 
that  the  colon  bacillus  finds  in  the  intestine  of  the  higher 
vertebrates  an  environment  better  suited  to  its  growth 
and  multiplication  than  any  other  which  occurs  in  nature. 
Houston  (i9O3a)  records  the  number  of  B.  coli  per 
gram  of  normal  human  feces  as  between  100,000,000 
and  1,000,000,000.  It  is  almost  certain  that  the  only 
way  in  which  large  numbers  of  these  organisms  gain 
access  to  natural  waters  is  by  pollution  with  the  domestic 


The  Significance  of  B.  Coli  in    Water.         121 

industrial,  and  agricultural  wastes  of  human  life.  If 
pollution  has  been  recent,  colon  bacilli  will  be  found  in 
comparative  abundance.  If  pollution  has  been  remote 
the  number  of  colon  bacilli  will  be  small,  since  there  is 
good  evidence  that  the  majority  of  intestinal  bacteria  die 
out  in  water.  If  derived  from  cereals  or  the  intestines 
of  wild  animals,  the  number  will  be  insignificant  except 
in  the  vicinity  of  great  grain-fields  or  where  the  water 
receives  refuse  from  grist-mills,  tanneries,  dairies,  or 
lactic-acid  factories. 

The  first  recognition  of  the  necessity  for  a  quantita- 
tive estimation  of  colon  bacilli  in  water  we  owe  to  Dr. 
Smith,  who  in  1892  (Smith,  i893a)  outlined  a  plan  for  a 
study  to  be  made  by  the  New  York  Board  of  Health  on 
the  Mohawk  and  Hudson  Rivers.  Burri  (Burri,  1895) 
pointed  out  that  the  use  of  so  large  a  sample  as  a  liter  for 
examination  would  lead  to  the  condemnation  of  many 
good  waters.  Freudenreich  (Freudenreich,  1895)  a* 
the  same  time  indicated  the  necessity  for  taking  into 
account  the  number  of  colon  bacilli  present.  He  recorded 
the  isolation  of  the  organism  from  unpolluted  wells, 
when  as  large  a  quantity  of  water  as  100  c.c.  was  used, 
and  concluded  that  it  was  entirely  absent  only  from 
waters  of  great  purity  and  present  in  large  numbers  only 
in  cases  of  high  pollution.  This  author  also  quoted 
Miquel  as  having  found  colon  bacilli  in  almost  every 
sample  of  drinking-water  if  only  a  sufficient  portion  were 
taken  for  analysis. 


122  Elements  of  Water  Bacteriology. 

The  practical  results  of  the  application  of  the  colon 
test  from  this  standpoint  have  proved  of  the  highest  value. 
As  originally  outlined  by  Dr.  Smith,  it  consisted  in  the 
inoculation  of  a  series  of  dextrose  tubes  with  small  por- 
tions of  water,  tenths  or  hundredths  of  the  cubic  centi- 
meter. It  was  first  used  by  Brown  (Brown,  1893)  in 
1892  for  the  New  York  State  Board  of  Health,  and 
showed  from  22  to  92  fecal  bacteria  per  c.c.  in  the  water 
of  the  Hudson  River  at  the  Albany  intake,  and  from  3  to 
49  at  various  points  in  the  Mohawk  River  between  Amster- 
dam and  Schenectady.  In  some  previous  work  at  St. 
Louis,  the  colon  bacilli  in  the  Mississippi  River  were 
found  to  vary  from  3  to  7  per  c.c. 

Hammerl  (Hammerl,  1897)  used  the  presence  of  Bacil- 
lus coli  as  a  criterion  of  self-purification  in  the  river 
Mur.  He  considered,  in  spite  of  the  position  taken  by 
Kruse,  that  when  a  water  contained  large  numbers  of 
colon  bacilli,  as  well  as  an  excess  of  bacteria  in  general, 
it  might  be  considered  to  be  contaminated  by  human  or 
animal  excrement.  As,  however,  the  organism  would 
naturally  be  present  in  large  quantities  of  such  a  water 
as  that  of  the  Mur,  he  used  no  enrichment  process,  but 
made  plate  cultures  direct;  he  defined  the  B.  coli  as  a 
small  bacillus,  non-motile  or  but  feebly  motile,  growing 
rapidly  at  37°  C.,  coagulating  milk  and  forming  gas 
in  sugar  media.  In  general,  Hammerl  failed  to  find 
colon  bacilli  in  the  river  by  this  method,  except  immedi- 
ately below  the  various  towns  situated  upon  it;  at  these 


The  Significance  of  B.  Coli  in    Water.          123 

points  of  pollution  he  discovered  a  few  colon  colonies 
upon  his  plates,  not  more  than  4  to  6  per  c.c.  of  the  water. 
He  concluded  that  "the  Bacterium  coli,  even  when  it  is 
added  to  a  stream  in  great  numbers,  under  certain  circum- 
stances disappears  very  rapidly,  so  that  it  can  no  longer 
be  detected  in  the  examination  of  small  portions  of  the 
water."  It  should  be  noted  that  Hammerl's  method 
was  much  less  delicate  than  the  use  of  the  dextrose  tube 
for  preliminary  incubation. 

The  most  important  work  upon  the  distribution  of  B. 
coli  has  been  that  carried  out  in  England  by  the  bacteri- 
ologists of  the  local  government  board,  by  Dr.  Houston 
in  particular.  This  investigator  (Houston,  1898;  Houston, 
i8g9a;  Houston,  i9ooa)  made  an  elaborate  series  of  exam- 
inations of  soils  from  various  sources  to  see  whether  the 
microbes  considered  to  be  characteristic  of  sewage  could 
gain  access  to  water  from  surface  washings  free  from 
human  contamination.  In  the  three  papers  published 
on  this  subject  the  examination  of  46  soils  was  recorded. 
In  only  10  of  the  samples  was  B.  coli  found,  and  of  these 
10,  9  were  obviously  polluted,  being  derived  from  sewage 
fields,  freshly  manured  land,  or  the  mud-banks  of  sewage- 
polluted  rivers.  The  author  finally  concluded  that  "as  a 
matter  of  actual  observation,  the  relative  abundance  of  B. 
coli  in  pure  and  impure  substances  is  so  amazingly  dif- 
ferent as  to  lead  us  to  suspect  that  not  only  does  B.  coli 
not  flourish  in  nature  under  ordinary  conditions,  but  that 
it  tends  to  even  lose  its  vitality  and  die. "  "In  brief,  I  am 


124  Elements  of  Water  Bacteriology. 

strongly  of  opinion  that  the  presence  of  B.  coli  in  any  num- 
ber, whether  in  soil  or  in  water,  implies  recent  pollution  of, 
animal  sort. "  Pakes  (Pakes,  1900)  stated  on  the  strength 
of  an  examination  of  "about  300  different  samples  of 
water,"  no  particulars  being  published,  that  water  from 
a  deep  well  should  not  contain  B.  coli  at  all,  but  that  water 
from  other  sources  need  not  be  condemned  unless  the 
organism  was  found  in  20  c.c.  or  less.  When  colon  bacilli 
were  found  only  in  greater  quantities  than  100  c.c.,  the 
water  might  be  considered  as  probably  safe.  Horrocks 
(Horrocks,  1901),  after  a  general  review  of  English  prac- 
tice, concluded  that  "when  a  water-supply  has  been 
recently  polluted  with  sewage,  even  in  a  dilution  of  one  in 
one  hundred  thousand,  it  is  quite  easy  to  isolate  the  B.  coli 
from  i  c.c.  of  the  water."  "I  would  say  that  a  water 
which  contained  B.  coli  so  sparingly  that  200  c.c.  required 
to  be  tested  in  order  to  find  it  had  probably  been  polluted 
with  sewage,  but  the  contamination  was  not  of  recent 
date."  Chick  (Chick,  1900)  found  6100  colon  bacilli  per 
c.c.  in  the  Manchester  ship  canal,  55  to  190  in  the  pol- 
luted River  Severn,  and  numbers  up  to  65,000  per  gram 
in  roadside  mud.  On  the  other  hand,  of  38  unpolluted 
streams  and  rivulets,  31  gave  no  Bacillus  coli  and  the 
other  7  gave  i  per  c.c.  or  less.  The  Liverpool  tap  water, 
snow,  rain,  and  hail  showed  no  colon  bacilli. 

One  of  the  first  elaborate  applications  of  the  colon  test 
was  made  by  Jordan  in  the  examination  of  the  fate  of 
the  Chicago  sewage  in  the  Desplaines  and  Illinois  Rivers. 


The  Significance  of  B.  Coli  in    Water.         125 

At  one  time  Professor  Jordan  was  himself  somewhat 
sceptical  as  to  the  value  of  the  colon  test,  for  he  stated  in 
1890  (Jordan,  1890)  that  he  had  found,  "in  spring-water 
which  was  beyond  any  suspicion  of  contamination,  bac- 
teria which  in  form,  size,  growth  on  gelatin,  potato,  etc., 
were  indistinguishable  from  B.  coli  commune."  In  the 
Chicago  studies  of  self-purification  (Jordan,  1901)  the 
analyses  were  made  quantitative  by  the  examination  of 
numerous  measured  samples,  fractions  of  the  cubic  centi- 
meter; and  the  method  employed  was  enrichment,  either 
in  dextrose-broth  fermentation  tubes  or  in  phenol  broth, 
with  subsequent  plating  on  litmus  lactose  agar.  The 
cultures  isolated  were  tested  as  to  their  behavior  in  dextrose 
broth,  peptone  solution,  milk,  and  gelatin;  of  the  dextrose 
tubes  made  directly  from  the  water  all  were  considered 
positive  which  gave  more  than  20  per  cent  gas  in  the 
closed  arm,  with  an  appreciable  excess  of  hydrogen.  The 
results  were  very  significant.  In  fresh  sewage  a  positive 
result  was  obtained  about  one-third  of  the  time  in  one 
one-hundred-thousandth  of  a  cubic  centimeter  and  almost 
constantly  in  one  ten-thousandth  of  a  cubic  centimeter. 
The  Illinois  and  Michigan  canal  proved  almost  as  bad, 
giving  positive  results  on  seven  days  out  of  twenty-eight  in 
dilutions  of  one  in  one  hundred  thousand  and  on  twenty- 
eight  days  out  of  thirty-two  in  a  dilution  of  one  in  ten 
thousand.  At  Morris,  twenty-seven  miles  below  Lockport, 
where  the  canal  enters  the  bed  of  the  Desplaines  River,  and 
nine  miles  below  the  entrance  of  the  Kankakee,  the 


126 


Elements  of  Water  Bacteriology. 


principal  diluting  factor,  the  numbers  were  so  reduced  that 
positive  results  were  obtained  only  on  eleven  days  out  of 
twenty  in  one-thousandth  of  a  cubic  centimeter,  on  twenty 
days  out  of  thirty  in  one-hundredth  of  a  cubic  centimeter, 
and  on  twenty  days  out  of  twenty-three  in  one-tenth  of  a 
cubic  centimeter.  At  Averyville,  one  hundred  and  fifty- 
nine  miles  below  Chicago,  colon  bacilli  were  isolated  on 
only  four  days  out  of  twenty-seven  in  one-tenth  of  a  cubic 
centimeter,  and  on  thirteen  days  out  of  thirty-one  in  one 
cubic  centimeter.  A  comparison  with  certain  neighboring 
rivers  showed  this  to  be  about  the  normal  value  for  waters 
of  similar  character,  as  the  following  table  extracted  from 
Professor  Jordan's  paper  will  show. 


NUMBER  OF  B.  COLI  PRESENT  IN  CERTAIN  RIVER 

WATERS. 
(JORDAN,    igoi.) 


.1  C 

.C. 

I    C. 

c. 

Source  of  Sample. 

No.  Days 
Water 
Examined. 

No.  Days 
B.  Coli 
Found. 

No.  Days 
Water 
Examined. 

No.  Days 
B.  Coli 
Found. 

Illinois  River,  Averyville  . 
Mississippi  River,  Grafton 
Fox  River  

27 
34 

22 

4 

10 

2 

31 
35 
23 

13 

'     23 
6 

Sangamon  River  .... 
Missouri  River  

25 
32 

14 
13 

27 
3i 

21 
21 

These  results  harmonize  rather  closely  with  those  pre- 
viously recorded  by  Brown  and  Fuller,  and  indicate  that 
in  the  larger  rivers  where  the  proportionate  pollution  is 


The  Significance  of  B.  Coli  in    Water.         127 

not  extreme,  colon  bacilli  may  be  isolated  in  about  half 
the  i-c.c.  samples  examined.  Such  rivers  are  of  course 
inadmissible  as  sources  of  water-supply,  according  to 
modern  sanitary  standards,  unless  subjected  to  purifica- 
tion of  some  sort. 

More  recently  Hunnewell  and  one  of  us  (Winslow  and 
Hunnewell,  1902^  examined  a  considerable  series  of 
normal  waters  for  B.  coli,  testing  i  c.c.  from  each  by  the 
dextrose-broth  method  and  a  larger  portion  of  100  c.c.  by 
incubation  with  phenol  broth  as  described  in  Chapter  VI. 
The  samples  were  obtained  from  the  public  supplies  of 
Taunton,  Bo'ston,  Cambridge,  Braintree,  Brookline,  Need- 
ham,  and  Lynn  in  Massachusetts,  and  Newport,  R.  L, 
from  the  Sudbury  River,  from  the  ocean,  from  the  waters 
of  springs  bottled  for  the  market,  from  ponds,  pools  of 
rain  and  melted  snow,  springs,  brooks,  shallow  wells,  and 
driven  wells  in  various  towns  near  the  city  of  Boston.  For 
comparison  50  samples  of  polluted  waters  from  the  Charles, 
Mystic,  Neponset,  and  North  Rivers  were  examined.  The 
colon  bacillus  was  defined  as  outlined  in  Chapter  VT,  and 
organisms  which  lacked  the  power  to  reduce  nitrates  or  to 
form  indol  were  classed  in  the  "Paracolon  group."  The 
results  are  summarized  in  the  following  table: 


128 


Elements  of  Water  Bacteriology. 


PRESENCE  OF  B.  COLI  IN  POLLUTED  AND  UNPOLLUTED 

WATERS. 
(WINSLOW  AND   HUNNEWELL, 

Unpolluted   Waters. 


I  C.C. 

100  C.C. 

Samples  examined     .    . 

1^7 

JC7 

Dextrose  broth  positive    

40 

76 

I? 

•21 

Colon  group 

5" 

J  J 

Paracolon  group 

5" 

r 

B.  cloaca?  group         .... 

tr 

Streptococcus  group  ... 

•7 

IO 

Polluted   Waters. 


I  C.C. 

100  C.C. 

Samples  examined                                      . 

CO 

48 

Dextrose  broth  positive        .... 

ro 

77 

Lactose  plate  positive  

CO 

26 

Colon  group    

18 

4 

Paracolon  group 

6 

Streptococcus  group 

2C 

22 

B.  cloaca?    

I 

As  the  authors  pointed  out,  these  tables  indicate  that 
bacteria  capable  of  growth  at  the  body  temperature  and 
fermenting  dextrose  and  lactose  are  infrequently  found  in 
unpolluted  waters,  and  colon  bacilli  are  very  rarely  present. 
In  157  samples,  typical  colon  bacilli  were  found  only  5 
times  out  of  157,  in  i  c.c.  Lactose  fermenting  organisms 
appeared  in  only  8  per  cent  of  the  normal  samples  and  in 
100  per  cent  of  the  polluted  ones,  in  i  c.c.  Incidentally 
it  may  be  pointed  out  that  these  tables  well  illustrate  the 
dangers  of  overgrowths,  particularly  in  large  samples.  It 


The  Significance  of  B.   Coli  in    Water.       129 


is  clear  that  the  streptococci  had  killed  out  colon  bacilli, 
originally  present,  in  a  large  proportion  of  the  zoo-c.c. 
samples  of  polluted  waters  and  in  some  of  the  i-c.c.  samples, 
since,  in  so  many  cases,  gas  formation  was  followed  by 
the  isolation  of  the  streptococcus  alone. 

Clark  and  Gage  (1903)  have  published  the  results  of 
certain  studies  of  Massachusetts  ponds  which  indicate 
clearly  the  coincidence  of  the  distribution  of  B.  coli  in 
single  centimeter  samples  of  surface  waters,  with  actual 
sanitary  conditions.  They  show  also  the  slight  signifi- 
cance of  the  test  for  this  organism  in  larger  volumes  of 
water.  Almost  every  source  gave  some  positive  tests  in 
100  c.c.,  while  with  i-c.c.  samples  only  those  lakes  appear 
suspicious  which  are,  in  fact,  exposed  to  dangerous  pol- 
lution. 

DISTRIBUTION    OF   TOTAL   BACTERIA   AND    B.    COLI   IN 

SURFACE    WATERS. 
(CLARK  AND  GAGE,  1903.) 


Lake. 

Population  of 
Watershed  per 

Bacteria 

per  c.c. 

B.  coli 
Per  cent  positive  Tests. 

Square  Mile. 

I  C.C. 

100  C.C. 

I* 

1400 

612 

13-3 

33-° 

2 

356 

319 

3-5 

17.2 

3 

116 

io3 

0.0 

0.0 

4 

90 

170 

o.o 

14.0 

5 

62 

87 

o.o 

9.0 

6* 

60 

48 

2-3 

4-5 

7* 

So 

66 

4-6 

21.0 

8 

47 

133 

o.o 

9.0 

9 

42 

131 

0.0 

6.7 

10* 

40 

3i 

0.0 

6.2 

ii 

8 

28 

o.o 

7-7 

12 

42 

107 

0.0 

9-3 

*  Shores  used  for  pleasure  resorts. 


130 


Elements  of  Water  Bacteriology. 


Houston  (1905)  gives  the  following  table  which  may  be 
taken  as  another  fair  example  of  the  distribution  of  B. 
coli  in  small  streams  and  lakes.  Of  the  two  lakes  studied, 
Loch  Ericht  is  free  from  the  pollution  of  human  or  domes- 
ticated animals  while  Loch  Laggan  receives  some  drainage 
from  farm  lands;  both  are  of  large  size.  The  brook  and 
river  samples  were  collected  from  adjacent  streams. 

DISTRIBUTION  OF  B.  COLI  IN  SURFACE  WATERS. 

(HOUSTON,   1905.) 
Percentage  of  Samples  showing  B.  coli  in  each  Dilution. 


Dilution. 

+  .1  C.C. 

+  I.O  C.C. 
—    .1  C.C. 

+  10  c.c. 

—    I.  C.C. 

+  100  C.C. 
—    10  C.C. 

Not  in 
ibo  c.c. 

Brooks    and 

River      .    .    . 

7-7 

53.8 

34-6 

3-8 

.  .  . 

Loch  Laggan   . 

1.2 

33-° 

49.4 

16.4 

Loch  Ericht.    . 

I.O 

19.0 

80.0 

As  an  example  of  a  heavily  polluted  stream,  on  the  other 
hand,  the  following  table  on  page  131  maybe  cited.  It 
shows  in  a  striking  way  the  increase  of  B.  coli  in  the 
Thames  on  its  passage  through  London  and  its  progressive 
purification  below. 

The  river  at  the  lower  stations  in  this  table  was  con- 
siderably diluted  with  sea-water,  yet  it  showed  clearly 
its  large  proportion  of  sewage.  Normal  sea-water,  even  in 
the  neighborhood  of  the  shore,  shows  B.  coli  only  in  large 
samples.  Houston  (1904),  in  another  communication, 
reports  the  examination  of  168  samples  of  sea-water  near 
the  English  coast.  None  of  the  samples  showed  B.  coli 


UNIVERSITY  OF  CALIFORNIA 
DEPARTMENT  OF  CIVIL  ENGINEER! 
BERKELEY,  CALIFORNIA 

The  Significance  of  B.  Coli  in    Water.          131 

in  i  c.c.;  97  samples  gave  negative  results  in  10  c.c.;  45  in 
100  c.c.,  and  4  had  20  B.  coli  even  in  1000  c.c. 

B.  COLI  IN  THE  RIVER  THAMES  AT  VARIOUS  POINTS. 

(HOUSTON,  1904*-) 
Percentage  of  positive  results. 


+  10 

+  i 

+  .1 

+  .01 

+  .001 

+   .0001 

Place. 

+  10 

—  I 

—  ,i 

—  .01 

—  .001 

—  .0001 

—  .00001 

c.c. 

C.C. 

c.c. 

c.c. 

c.c. 

c.c. 

c.c. 

Sunbury   . 

70.6 

23-5 

5-9 

Hampton 

. 

iY.8 

64.7 

17.7 

5-9 

Barking  . 

.  .  . 

4.2 

45-8 

45-8 

4-2 

Crossness 

ii.  i 

27.7 

50.0 

II.  I 

Purfleet     .    \   ^ 

3-° 

9.1 

33-3 

39-i 

15-1 

Grays    .    . 

p 

2.8 

22.2 

41-7 

33-3 

Mucking  . 

30.8 

57-7 

"•5 

Chapman 

5-° 

45-° 

5°- 

.  .  . 

Barrow  Deep 

12.  O 

36.0 

40. 

12.  O 

With  ground-waters  the  story  is  the  same.  Even  in 
sources  of  excellent  quality  we  should  expect  to  find,  and 
we  do  sometimes  find,  colon  bacilli  in  large  volumes  of 
water.  Abba,  Orlandi,  and  Rondelli  (1899)  showed  by 
experiments  with  B.  prodigiosus  at  Turin  that  when 
bacteria  are  present  in  great  numbers  on  the  surface  of  the 
ground,  a  few  may  penetrate  for  a  considerable  distance 
and  ultimately  reach  the  sources  of  ground-waters.  The 
chance  that  disease  germs  could  survive  this  process  in  a 
soil  so  impervious  as  to  allow  colon  bacilli  to  appear  only 
in  large  samples  of  water,  is  however  infinitesimal. 

An  interesting  contribution  to  the  bacteriology  of  ground- 
waters  was  made  by  the  Massachusetts  State  Board  of 


132  Elements  of  Water  Bacteriology. 

Health  (Massachusetts  State  Board  of  Health,  1901)  in 
connection  with  the  examination  of  the  spring-waters 
bottled  for  sale  in  the  state.  Ninety-nine  springs  were 
included  in  this  study,  and  in  almost  every  instance  4 
samples  were  examined,  2  taken  directly  from  the  spring 
by  the  engineers  of  the  board  and  2  from  the  bottles  as 
delivered  for  sale  to  the  public.  In  the  water  of  one 
spring  B.  coli  was  found  twice,  once  in  a  sample  from 
the  spring  and  once  in  the  bottled  sample.  This  spring 
was  situated  in  woodland,  but  was  unprotected  from 
surface  drainage,  and  the  method  of  filling  bottles  sub- 
jected it  to  possible  contamination.  In  5  other  cases  B. 
coli  was  found  once  in  the  sample  from  the  spring;  all 
were  subject  to  pollution  from  dwellings  or  cultivated 
fields,  and  4  of  the  5  were  shown  to  be  highly  contam- 
inated, chemically.  In  7  other  cases  B.  coli  was  found 
in  the  bottled  samples  alone;  3  of  these  sources  were  of 
high  purity,  but  the  bottling  process  furnished  oppor- 
tunity for  contamination. 

Clark  and  Gage  (1903),  hi  the  examination  of  170 
samples  of  water  from  tubular  and  curb  wells  of  good 
quality  used  as  sources  of  water-supply,  found  B.  coli  only 
five  times,  once  in  one  cubic  centimeter  and  four  tim.es  in 
one  hundred  cubic  centimeters.  Horton  (1903),  from  a 
study  of  ground-waters  in  Ohio,  concluded  that  the 
presence  of  B.  coli  in  wells  and  springs  was  indicative  of 
serious  pollution;  of  37  waters  of  this  class  which  showed 
B.  coli,  27  had  a  history  of  typhoid  fever. 


The  Significance  of  B.  Coli  in   Water.        133 


Houston  (i9O3b)  makes  an  instructive  comparison  of 
some  more  or  less  polluted  shallow  wells  at  Chichester 
with  deep  ground-waters  of  high  quality  at  Tunbridge 
Wells.  The  following  table  shows  the  value  of  the  one- 
cubic-centimeter  sample  in  discriminating  between  good 
and  bad  waters : 

DISTRIBUTION   OF   B.    COLI    IN   GOOD   AND   BAD   WELL 

WATERS. 

(HOUSTON,  igo3b.) 

Percentage  of  Positive  Tests. 


Quantity  of  Water. 

Chichester  Shallow 
Wells. 

Tunbridge  Wells,  Deep 
Wells. 

100  c.c. 

10      C.C 

90 
80 

1 

I      C.C. 
O.  I  C.C. 

45 
20 

o 
o 

In  a  subsequent  investigation,  Houston  (1905)  exam- 
ined still  larger  samples  of  water  from  the  Tunbridge 
Wells  for  B.  coli:  49  samples  of  100  c.c.  each  showed  no 
B.  coli,  and  27  liter  samples  showed  B.  coli  only  once. 
Kaiser  (1905)  reports  an  interesting  correlation  between 
total  numbers  and  B.  coli  in  a  series  of  38  well  waters.  Of 
ii  wells  containing  over  200  bacteria  per  c.c.  90  per  cent 
showed  colon-like  organisms  in  liter  samples.  Of  12  wells 
containing  from  50  to  200  bacteria  per  c.c.  67  per  cent 
gave  colon-like  organisms;  of  26  wells  with  less  than  50 
bacteria  per  c.c.,  only  27  per  cent  showed  positive  results. 

One  of  the  most  important  applications  of  the  colon 


134 


Elements  of  Water  Bacteriology. 


test  is  in  the  control  of  the  operation  of  municipal  water 
filters.  It  has  been  used  for  this  purpose  for  ten  years  or 
more  at  Lawrence,  and  Fuller  laid  stress  upon  its  results 
in  his  classic  experiments  on  water  purification  in  the 
Ohio  valley.  At  Cincinnati  he  records  the  presence  of 
colon  bacilli  in  60  per  cent  of  the  i-c.c.  samples  from  the 
Ohio  River,  while  the  effluent  from  either  slow  sand  or 
mechanical  filters  gave  positive  results  only  half  the  time 
in  samples  of  50  c.c.  The  results  of  the  examinations 
carried  out  at  Lawrence  for  six  years  are  brought  together 
in  the  table  below  from  the  Annual  Reports  of  the  Massa- 
chusetts State  Board  of  Health. 

B.  COLI  IN  MERRIMAC  RIVER  AND  LAWRENCE  FILTER 

EFFLUENT. 


Merrimac  River,  Per 
cent  of  one  c.c., 
Samples  containing 

Merrimac  River, 
Number  B.  coli  per 

Filtered  Water,  Per 
cent  of  one  c.c., 
Sample  containing 

B.  coli. 

B.  coli. 

1900 

99-7 

87 

18.1 

1901 

* 

* 

* 

1902 

99.0 

73 

4.0 

1903 

99.0 

78 

4.2 

1904 

100.  0 

73 

8.0 

1905 

100.  0 

118 

4-7 

*  Not  given. 

Effluents  of  better  character  are  obtained  by  the  filtration 
of  less  polluted  streams,  though  the  per  cent  purification 
effected  is  not  so  great  as  at  Lawrence.  Thus,  at  Harris- 
burg,  Pa.,  with  one  cubic  centimeter  samples,  positive 
tests  were  obtained  72  per  cent  of  the  time  in  the  raw 


The  Significance  of  B.  Coli  in   Water.        135 

Susquehanna  River  water  and  in  less  than  3  per  cent 
of  the  samples  of  filtered  effluent  (Harrisburg,  1907). 
At  Washington  the  most  complete  slow-sand  filtration 
plant  yet  constructed  has  yielded  the  results  tabulated 
below,  for  which  we  are  indebted  to  the  courtesy  of 
Mr.  F.  F.  Longley. 


B.  COLI  IN   POTOMAC  RIVER  AND  WASHINGTON  FILTER 
EFFLUENT. 


Dalecarlia  Reservoir  Inlet. 

Filtered-  Water  Reservoir 
Outlet. 

Samples. 

Samples. 

Number 

Number 

Tested. 

Tested. 

10  C.C. 

I  C.C. 

10  C.C. 

I  C.C. 

1906. 

February     . 

15 

5 

3 

24 

0 

o 

March      .    . 

24 

12 

3 

27 

o 

0 

April    .    .    . 

18 

9 

6 

25 

I 

o 

May     .    .    . 

25 

3 

i 

27 

o 

o 

June     .    .    . 

26 

9 

8 

26 

0 

0 

July      .    .    . 

20 

8 

9 

21 

I 

0 

August     .    . 

26 

21 

14 

27 

I 

I 

September  . 

10 

4 

i 

25 

2 

o 

October  .    . 

10 

3 

2 

27 

I 

o 

November  . 

8 

3 

0 

25 

2 

0 

December   . 

9 

4 

4 

24 

2 

2 

1907. 

January  .    . 

9 

5 

3 

26 

3 

3 

February.    . 

8 

2 

2 

23 

0 

0 

March     .    . 

8 

7 

4 

26 

0 

0 

April    .    .    . 

9 

4 

I 

26 

I 

o 

May     .    .    . 

23 

21 

15 

26 

o 

0 

June     ... 

25 

20 

17 

2S 

0 

o 

July     .    .    . 

26 

II 

8 

26 

0 

0 

August     .    . 

27 

13 

8 

27 

0 

0 

September  . 

24 

15 

13 

25 

I 

0 

136 


Elements  of  Water  Bacteriology. 


It  must  be  remembered  that  in  the  Washington  plant 
nitration  is  supplemented  by  thorough  sedimentation,, 
preliminary  and  subsequent.  The  entire  credit  for  the 
good  effluent  obtained  is  not  therefore  due  to  the  niters. 
At  Lawrence  it  has  been  shown  that  removal  of  colon 
bacilli  in  storage  reservoirs  and  pipe  systems  may  be 
considerable.  The  figures  obtained  in  1900  at  various 
points  in  the  distribution  system  may  be  cited  as  an 
example. 

PERCENTAGE    OF      SAMPLES    OF    WATER    CONTAINING 

B.   COLL 

Lawrence  Experiment  Station  (Massachusetts  State  Board  of  Health,   1901.) 


Effluent 
of  Filter. 

Outlet  of 
Reservoir. 

Tap, 
City  Hall. 

Tap.Experi 
ment 
Station. 

In  i  c.c  . 

18.14 

8.0 

4.07 

1.87 

In  100  c.c  

38.12 

23-3° 

iS-54 

15-54 

In  regard  to  the  proportion  of  positive  colon  tests  per- 
missible in  a  filter  effluent,  Clark  and  Gage  (Clark  and 
Gage,  1900)  reported  some  specially  instructive  obser- 
vations made  when  certain  of  the  underdrains  of  the 
Lawrence  filter  were  relaid  in  the  autumn  of  1898.  In 
doing  this  work  the  sand  on  some  of  the  beds  was  seriously 
disturbed;  and  in  December,  after  the  work  was  com- 
pleted, B.  coli  was  found  in  i  c.c.  of  the  filtered  effluent 
in  72  per  cent  of  the  samples  examined.  In  January  and 
February  the  organisms  were  found  in  54  per  cent  and  62 
per  cent  of  the  samples,  respectively,  while  in  March  the 


The  Significance  of  B.  Coli  in    Water.        137 

number  fell  to  a  normal  value  of  8  per  cent.  Correspond- 
ing to  this  excess  of  B.  coli  in  the  city  water,  there  were  12 
cases  of  typhoid  fever  in  December,  59  cases  in  January, 
12  in  February,  and  9  in  March,  all  during  the  early  part 
of  the  month.  The  authors  conclude  that  "  when  filtering 
a  river-water  as  polluted  as  that  of  the  Merrimac,  it  is 
safe  to  assume  that  when  B.  coli  is  found  only  infre- 
quently in  i  c.c.  of  the  effluent,  the  typhoid  germs,  neces- 
sarily fewer  in  number  and  more  easily  removed  by  the 
filter,  have  been  eliminated  from  the  water." 

The  results  of  the  daily  tests  carried  out  at  municipal 
filter  plants  are  frequently  expressed  in  monthly  or  yearly 
averages,  as  in  some  of  the  cases  quoted  above.  It  must 
be  remembered,  however,  that  averages  of  this  sort  are 
accepted  only  by  courtesy  and  with  the  implied  assump- 
tion that  conditions  are  approximately  constant  during 
the  period  averaged.  When  it  is  said  that  an  acceptable 
effluent  may  show  B.  coli  in  three  or  four  per  cent  of  the 
samples  tested,  the  statement  is  true  only  for  a  series  of 
samples  collected  and  examined  at  the  same  time.  If  in 
a  given  month  3  per  cent  of  the  i-c.c.  samples  tested  show 
B.  coli,  the  effluent  may  or  may  not  be  safe.  If  on  each 
of  20  days  3  B.  coli  or  thereabouts  were  present  in  100  c.c. 
of  the  water,  it  is  probably  a  safe  one.  If  on  19  days  no 
B.  coli  were  present,  and  on  the  twentieth  day  100  c.c. 
showed  60  B.  coli,  the  average  result  would  be  the  same, 
but  the  water  on  one  day  was  of  a  dangerous  character. 
With  properly  managed  filter  plants  marked  variations  do 


138  Elements  of  Water  Bacteriology. 

not  occur  from  day  to  day  and  average  results  are  gen- 
erally reliable.  It  is  wholly  misleading,  however,  to  com- 
pare such  results  with  the  average  examinations  of  an 
unfiltered  surface  water.  With  surface  waters  daily  vari- 
ations are  the  rule  and  a  low  monthly  average  of  colon 
tests  may  include  and  cover  up  dangerous  and  significant 
high  numbers  at  particular  periods. 

The  general  results  of  the  studies  of  the  colon  tests 
which  have  now  been  carried  out  in  great  numbers  all 
over  the  world  may  be  summarized  by  a  few  further 
citations. 

In  America  the  fact  that  the  number  of  colon  bacilli  in 
a  water  measures  the  degree  of  its  pollution  is  now  univer- 
sally accepted.  The  same  conclusion  has  been  estab- 
lished in  England  by  the  elaborate  investigations  of 
Houston  and  his  pupils.  Savage,  for  example,  concluded 
(Savage,  1902)  from  a  study  of  a  large  number  of  water 
supplies  in  Wales,  that  even  in  surface  waters,  exposed 
to  animal  contamination  from  adjacent  grazing  grounds, 
B.  coli  is  not  present  in  2  c.c.  unless  other  pollution  is 
present.  In  a  more  recent  review  of  the  whole  subject, 
the  same  author  (Savage,  1906)  concludes  that  "there  is 
no  evidence  or  observations  which  have  ever  shown  that 
B.  coli,  reasonably  defined,. is  present  in  any  numbers  in 
sources  which  have  not  been  exposed  to  some  form  of 
fecal  contamination." 

In  Germany,  Petruschky  and  Pusch  (Petruschky  and 
Pusch,  1903)  examined  a  considerable  series  of  waters 


The  Significance  of  B.  Coli  in    Water.        139 

from  different  sources  by  incubating  measured  samples 
with  equal  amounts  of  nutrient  broth  and  isolating  upon 
agar.  In  45  samples  of  well-waters  they  found  B.  coli 
7  times  in  .01  c.c.,  9  times  in  .1  c.c.,  and  7  times  in  i  c.c. 
In  the  other  22  cases  it  could  not  be  found  in  i  c.c.  and  in 
4  cases  not  in  100  c.c.  One  sample  showed  it  only  in  600 
c.c.  and  i  not  in  750  c.c.  Of  29  river-waters,  only  2  failed 
to  give  positive  results  in  .1  c.c.  and  14  showed  B.  coli  in 
.001  of  a  c.c.  or  less.  In  sewage  the  number  varied  from 
i  to  1,000,000  per  c.c.  The  authors  conclude  that  a 
quantitative  estimation  of  the  B.  coli  content  furnishes  a 
good  measure  of  the  fecal  pollution  of  water.  Some  of 
the  best  French  bacteriologists  have  recently  come  to  a 
similar  conclusion.  Gautie  (1905)  holds  that  the  quanti- 
tative determination  of  B.  coli  is  of  the  highest  importance 
in  water  analysis;  and  Vincent  (1905),  in  an  excellent 
review  of  the  subject,  gives  strong  reasons  for  maintaining 
the  same  position.  He  finds  B.  coli  absent  from  spring 
and  well-waters  of  good  quality  and  present  in  polluted 
water  in  proportion  to  its  pollution.  A  number  of  French 
rivers  showed  numbers  of  B.  coli  varying  from  i  to  no 
per  c.c.  He  concludes  finally  that  water  containing  B. 
coli  in  .1  to  i. o  c.c.  is  unfit  to  drink,  while  if  the  organism 
is  found  in  i.o  to  10.0  c.c.  it  is  of  doubtful  quality. 

Altogether  the  evidence  is  quite  conclusive  that  the 
absence  of  B.  coli  demonstrates  the  harmlessness  of  a 
water  as  far  as  bacteriology  can  prove  it.  That  when 
present,  its  numbers  form  a  reasonably  close  index  of 


140  Elements  of  Water  Bacteriology. 

the  amount  of  pollution,  the  authors  above  quoted  have 
proved  beyond  reasonable  cavil.  It  may  safely  be  said 
that  when  the  colon  bacillus,  as  denned  by  the  tests  above, 
is  found  in  such  abundance  as  to  be  isolated  in  a  large 
proportion  of  cases  from  i  c.c.  of  water,  it  is  reasonable 
proof  of  the  presence  of  serious  pollution. 


CHAPTER   VIII. 

PRESUMPTIVE  TESTS  FOR  B.  COLI. 

THE  isolation  and  identification  of  B.  coli  by  the 
methods  which  have  been  described  is  a  time-consuming 
and  laborious  operation,  and  one  sometimes  difficult  to 
apply  in  the  practical  supervision  of  a  water-supply. 

Hence  many  investigators  have  attempted  to  devise 
tests  which  might  be  easily  and  quickly  carried  out,  and 
which  would  yet  give  a  fairly  correct  idea  as  to  the 
existence  of  pollution.  Such  tests  are  spoken  of  as 
"presumptive  tests." 

The  medium  which  was  first  urged  for  a  rapid  presump- 
tive test  was  dextrose  broth;  and  this  method  gained 
considerable  acceptance  five  years  ago.  Its  underlying 
principle  is  that  B.  coli  develops  rapidly  in  dextrose  broth 
with  gas  formation  of  from  25  to  70  per  cent  of  the  capac- 
ity of  the  closed  arm  of  the  fermentation  tube.  Of  this 
gas  approximately  one-third  is  carbon  dioxide  and  two- 
thirds  hydrogen,  that  is,  as  the  gas  formula  is  generally 

A          H  2 

expressed,    =  — . 

C02      i 

In  testing  a  water  by  this  method  a  series  of  samples, 
in  suitable  dilution,  .001,  .01,  .1,  i.o,  or  ioc.c.,  is  added 

141 


142  Elements  of  Water  Bacteriology. 

directly  to  the  dextrose-broth  tubes  and  incubated  for 
twenty-four  hours  at  37  degrees. 

On  measurement  of  the  gas,  if  the  results  above  given 
are  obtained,  the  reaction  is  considered  typical.  If  the 
amount  of  gas  is  between  10  and  25  per  cent  or  more  than 
70  per  cent,  or  the  percentage  of  carbon  dioxide  is  greater 
than  40,  the  reaction  is  considered  atypical.  If  no  gas 
forms,  or  less  than  10  per  cent,  the  test  is  called 
negative. 

In  recent  years,  Irons  (Irons,  1901)  was  perhaps  the 
first  to  call  attention  to  the  value  of  this  method,  v  stat- 
ing that  "when  the  dextrose  tube  yields  approximately 
33  per  cent  of  CO2,  Bacillus  coli  communis  is  almost 
invariably  present."  In  the  next  year  the  reliability  of 
the  fermentation  test  as  an  indication  of  B.  coli  was 
worked  out  by  Gage  (Gage,  1902)  as  given  in  the  follow- 
ing table: 


I  C.C. 

IOO  C.C. 

Number  of  samples  tested    

<I72 

1-271; 

Number  giving  preliminary  fermentation 

IO"?6 

474 

Per  cent  of  latter  proved  to  contain  coli     

70 

71 

Whipple  (Whipple,  1903)  examined  a  large  number 
of  surface-water  supplies  by  this  "presumptive  test" 
and  obtained  striking  results,  shown  in  the  following 
table.  The  waters  are  arranged  in  six  groups  according 
to  the  results  of  sanitary  inspection,  Group  I  including 
waters  collected  from  almost  uninhabitated  watersheds, 


Presumptive   Tests  for  B.   Coli. 


143 


and  Group  VI  waters  too  much  polluted  to  be  safely  used 
for  domestic  purposes. 


PERCENTAGE    OF    SAMPLES    OF    WATERS    OF    VARIOUS 
SANITARY    GRADES    GIVING    POSITIVE    TESTS    FOR    B. 
COLI  WHEN  DIFFERENT  AMOUNTS  WERE  EXAMINED. 
(WHIPPLE,  1903.) 


Group  . 

O.I    C.C. 

I.O  C.C. 

IO  C.C. 

IOO  C.C. 

500  c.c. 

I    
II    

0.0 

ir   o 

3-5 

7    3 

20.8 
ic   o 

50.0 
60.  o 

50.0 
60.0 

Ill    

o  o 

7-  o 

SO.  O 

SO.O 

60.0 

IV    

4  O 

6.8 

41  .  7 

67.0 

7C..O 

v 

50 

I  3    O 

71?    o 

IOO    O 

IOO    O 

VI    „   .    .    .    . 

5-° 

2O.  2 

75-o 

80.0 

100.0 

In  view  of  these  results  Whipple  suggested  the  following 
provisional  scheme  of  interpretation: 


Presumptive  Test  for  Bacillus  Coli. 


Sanitary  Quality. 

O.OI 
C.C. 

O.I   C.C. 

I.O  C.C. 

IO.O 
C.C. 

IOO 
C.C. 

Safe                          .  ".•   ; 

o 

o 

o 

o 

4- 

Reasonably  safe 

o 

o 

o 

4- 

4. 

Questionable  

o 

o 

4- 

4- 

4- 

Probably  unsafe     

o 

4- 

4- 

4- 

4- 

Unsafe     . 

4_ 

4- 

4. 

4. 

4. 

It  is  undoubtedly  true  that  a  negative  presumptive 
test  is  generally  obtained  with  unpolluted  waters.  For  ex- 
ample, in  a  study  previously  cited,  Winslow  and  Nibecker 
(1903)  reported  that  of  775  dextrose-broth  tubes  inocu- 
lated from  259  unpolluted  sources,  only  41  showed  gas. 


144  Elements  of  Water  Bacteriology. 

On  the  other  hand,  it  is  equally  true  that  in  a  large  pro- 
portion of  cases  colon  bacilli  are  isolated  from  positive 
dextrose-broth  tubes.  Longley  and  Baton  (1907)  in  the 
examination  of  3553  samples  of  Potomac  water  obtained 
positive  tests  794  times,  while  B.  coli  was  actually  present 
529  times;  67  per  cent  of  the  presumptive  tests  were 
therefore  correct.  Gage  (1902),  in  the  Massachusetts 
work  cited  above,  found  that  70  per  cent  of  his  fermented 
dextrose  tubes  contained  B.  coli. 

The  work  of  recent  years  has  made  it  clear,  however, 
that  both  the  coincidence  of  negative  presumptive  tests 
with  the  absence  of  B.  coli  and  the  general  coinci- 
dence of  positive  presumptive  tests  with  the  presence  of 
B.  coli,  are  open  to  disastrous  exceptions.  In  the  study 
of  the  samples,  tabulated  on  page  142,  the  presumptive 
test  closely  coincided  with  sanitary  conditions.  In  the 
Kennebec  River,  too,  Whipple  found  a  close  corre- 
spondence between  the  results  of  the  presumptive 
test  and  the  complete  isolation  of  B.  coli  (Whipple, 
1907).  With  other  waters,  however,  discordant  results 
have  been  reported.  Stoughton  (1905),  in  a  study  of  the 
New  York  Supply,  showed  that  B.  coli  could  frequently 
be  isolated  when  the  presumptive  test  was  negative. 
Fuller  and  Ferguson's  (1905)  results  at  Indianapolis 
and  those  of  many  other  observers  have  led  to  the  same 
conclusion.  With  heavily  polluted  waters  the  presump- 
tive test  breaks  down  entirely.  Gas  production  may  be 
absent  or  atypical  in  a  large  proportion  of  tubes  inocu- 


Presumptive  Tests  for  B.  Coli.  145 

lated  with  water  containing  many  streptococci  or  other 
sewage  forms. 

On  the  other  hand,  with  some  waters,  positive  presump- 
tive tests  may  be  obtained  when  colon  bacilli  are  not 
present.  According  to  Clark  and  Gage  (1903)  there 
are  fifty-eight  well-described  species  of  bacteria  which 
give  the  presumptive  test  in  dextrose-broth,  of  which 
23  are  widely  separated  from  the  B.  coli  group.  A 
recent  unpublished  investigation  by  Winslow  and  Phelps, 
indicates  that  the  result  of  the  dextrose  broth  test  is 
markedly  influenced  by  the  factor  of  temperature.  Their 
work  consisted  in  the  examination  of  185  samples  of 
water  from  90  different  sources,  ponds,  brooks,  pools, 
wells  and  springs  in  five  different  states  (Maine,  New 
Hampshire,  Massachusetts,  Michigan  and  Virginia)  at 
three  different  seasons  of  the  year.  All  the  waters  exam- 
ined were,  as  far  as  could  be  determined,  free  from  spe- 
cific pollution,  although  washings  from  roads  or  pasture- 
land  might  have  had  access  to  some  of  them.  Most 
of  the  sources  were  undoubtedly  unpolluted,  and  the 
examination  of  119  samples  for  B.  coli  yielded  only  12 
positive  results.  The  presumptive  test,  however,  was 
obtained  in  a  large  proportion  of  the  cases,  and  much 
more  often  in  summer  than  in  winter  or  spring,  as 
indicated  in  the  table  on  page  146. 


146 


Elements  of  Water  Bacteriology. 


DEXTROSE  BROTH  FERMENTATION  IN  185  SAMPLES  OF 
NORMAL  WATERS   AT  DIFFERENT   SEASONS. 

(WINSLOW  AND  PHELPS.) 
PERCENTAGE  OF  POSITIVE  RESULTS. 


Summer 
1906. 

Winter. 

Spring. 

Summer 
1907. 

Framingham,  Mass.    .    .    . 
Ann  Arbor  Mich. 

87 

Qf 

62 

47 

23 

57 

Exeter,  N.  H  

82 

IO 

44 

c;o 

Richmond.  Va  

14 

14 

Mt.  Desert,  Me  

QC 

All  Stations 

01 

•37 

2C 

^4 

The  Ann  Arbor  waters  in  this  series  included  a  number 
of  driven  wells,  and  the  Mt.  Desert  sources  were  mountain 
brooks  and  ponds  of  the  highest  sanitary  quality. 

A  new  presumptive  test  has  recently  been  suggested 
by  Jackson  (1906),  which  promises  more  satisfactory 
results  than  dextrose  broth.  MacConkey  (1900)  long  ago 
suggested  the  use  of  media  containing  bile  salts  (sodium 
taurocholate)  for  the  differentiation  of  B.  coli  and  B.  typhi, 
and  bile-salts  media  have  been  used  by  various  English 
observers  (MacConkey,  1901;  MacConkey  and  Hill,  1901) 
for  the  isolation  of  sewage  bacteria.  Jackson  studied 
the  action  of  various  bile  media  and  showed  their  selec- 
tive inhibitory  action  in  the  striking  table  quoted  on  page 
147.  His  important  contribution  to  the  subject,  however, 
was  the  discovery  that  ox  bile  itself  could  be  used  as  a 
culture  medium,  and  that  it  was  easier  to  prepare,  cheaper 
and  more  effective  than  combinations  of  meat  infusion 
with  the  purified  bile  salts. 


Presumptive  Tests  for  B.  Coli. 


147 


SELECTIVE   ACTION   OF   BILE   SALTS. 
(JACKSON,   1906.) 


Bacteru 

i  per  c.c. 

Uncon- 

taminated 
Well. 

Contami- 
nated 
Pond. 

Suspen- 
sion of 
Feces. 

Suspen- 
sion of 
Feces. 

Gelatin   20° 

Q2O 

27OO 

•3  en  OOO 

OOO  OOO 

Agar   37° 

2^ 

1  7O 

A  CO  OOO 

OOO  OOO 

Bile  asar  *  *7° 

14. 

A-l 

•?oo  ooo 

OOO  OOO 

Lactose  bile  agar,*  37°  .    . 
Lactose  bile  agar,*  37°  .    . 
Bile  aear,  37°   . 

0 
0 

o 

25 
3 

250,000 
250,000 

60,000 

675,000 

600,000 
900,000 

*  Bile  diluted,  1:1. 

Jackson  therefore  suggested  the  use  of  fresh  ox  bile 
containing  i  per  cent  of  lactose  as  a  presumptive  test 
instead  of  dextrose  broth.  In  particular  he  hoped  that 
this  medium  would  be  free  to  a  great  degree  from  the 
negative  results  due  to  overgrowths  in  polluted  waters. 
He  reported  275  examinations  of  badly  contaminated 
waters,  in  which  65  per  cent  of  the  samples  failed  to 
give  the  dextrose-presumptive  test,  and  only  10  per  cent 
failed  to  show  gas  in  lactose  bile.  In  a  more  recent 
communication,  Jackson  (1907)  reports  that  in  the  ex- 
amination of  5000  samples  of  water  at  the  Mt.  Prospect 
Laboratory,  the  bile  medium  has  proved  uniformly  satis- 
factory. He  recommends  incubation  for  72  hours,  results 
being  commonly  obtained,  however,  after  48  hours;  and  he 
considers  any  tube  showing  25  per  cent  gas  as  positive. 
In  a  series  of  examinations  recently  carried  out  at  the  In- 
stitute of  Technology,  16  per  cent  of  the  positive  tubes 


148 


Elements  of  Water  Bacteriology. 


showed  gas  in  24  hours,  73  per  cent  after  48  hours, 
and  the  remaining  27  per  cent  only  after  72  hours,  so 
that  the  72-hour  period  is  frequently  necessary.  Sawin 
(1907)  reports  comparative  results  with  dextrose  broth 
and  bile  on  different  classes  of  waters,  the  most  striking 

COMPARATIVE    PRESUMPTIVE    TESTS    WITH    DEXTROSE 
BROTH   AND   LACTOSE   BILE. 
(SAWIN,  1907.) 


Source. 

Percentage  of  Samples  Giving  Positive  Tests 
for  B.  Coli. 

Dextrose  Broth. 

Lactose  Bile. 

O.I   C.C. 

I.O  C.C. 

IO.O 
C.C. 

O.I    C.C. 

I.O  C.C. 

IO.O 
C.C. 

i  Deep  wells           .    . 

0. 
0. 

15  -o 

5-2 

IO.O 
10.  0 

o. 

IO.O 

0. 
I.O 
IO.O 

15-7 

5-o 
10.5 

IO.O 

15.0 

0. 
IO.O 

15-° 

21.  O 
4O.O 
26.0 

5-° 
35-o 

0. 
0. 

5-o 
5-2 

IO.O 
IO.O 

00. 
0. 

0. 
0. 
0. 

S-2 
5-° 
10.5 

0.0 

5-° 

0. 

6.0 

i5-° 
31.0 

i5-o 
50.0 
15.0 
30.0 

2  Shallow  wells      .    .    . 
3  Lake    

4  Lake    

s  Lake 

6  Lake    . 

7  Lake    

8  Lake    

Average,  Nos.  3,  4,  5, 
6,  7,  8 

8-3 

47.0 
26.3 
36.8 

II.  O 

72.2 

37-6 

55-i 

23.6 

55-5 
73-7 
68.9 

5-o 

50.0 

30.0 
40.0 

4-3 

75-° 
90.0 

73-5 

26.0 

84.2 
85.0 
78.1 

9  River   

10  River 

ii  River           '             . 

Average,  Nos.  9,  10,  n 
12  Brook 

36.7 

47-7 
50.0 
25.0 

5S-i 

63.2 

73-7 
25.0 

66.0 

72.2 
78.9 

8.2 

40.0 

60.0 
84.2 
87-5 

79-4 

90.0 
90.0 
93-7 

82.4 

84.2 
90.0 
81.2 

13  Drainage 

1  4  Sewage     . 

Average,  Nos  12,  13,  14 

40.9 

53-9 

53-i 

77.2 

91.2 

85.1 

Presumptive   Tests  for  B.   Coli. 


149 


of  which  are  tabulated  on  page  148.  It  is  clear  that  the 
bile  medium  is  superior  to  dextrose  broth  for  the  more 
polluted  waters. 

It  has  been  pointed  out  in  Chapter  VI  that  the  lactose- 
bile  medium  is  inferior  to  dextrose  broth  as  a  preliminary 
enrichment  medium  for  the  full  isolation  of  B.  coli,  from 
the  fact  that  it  occasionally  prevents  the  growth  of  B. 
coli  which  may  be  isolated  by  the  dextrose  method.  As 
a  presumptive  test,  however,  it  is  far  superior  to  dextrose 
broth,  giving  a  higher  proportion  of  positive  tests  with 
polluted  waters  and  a  lower  proportion  of  erroneous 
positive  tests  with  waters  of  good  quality.  In  a  recent 
examination  of  176  surface  waters  in  eastern  Massachu- 
setts, carried  out  under  our  direction,  B.  coli  was  isolated 
70  times.  The  dextrose-broth  test  was  positive  120 
times,  an  error  of  70  per  cent;  while  the  bile  test,  alone, 
was  positive  78  times,  an  error  of  only  n  per  cent.  The 
tabulated  results  of  these  experiments  indicate  fairly  the 
merits  of  the  bile  medium  for  preliminary  enrichment  and 
as  a  presumptive  test. 

PRELIMINARY  AND  COMPLETE  RESULTS  OF  DEXTROSE 
BROTH   AND    BILE   TESTS.     176    SURFACE    WATERS. 


Preliminary  Positive 
Results. 
(Gas  Formation.) 

Final  Positive  Results. 
(B.  Coli). 

Dextrose  broth  .... 
Lactose  bile    

I2O 

78 

70 
64 

150  Elements  of  Water  Bacteriology. 

It  is  certain  that  the  bile  medium  sometimes  shows  gas 
when  typical  B.  coli  are  absent;  it  is  certain  that  it  somer 
times  gives  entirely  negative  results  when  B.  coli  may  be 
isolated  in  other  ways.  Neither  of  these  errors  is  com- 
monly of  great  magnitude,  however,  and  in  general  the 
lactose  bile  results  correspond  fairly  well  with  those 
obtained  by  the  complete  isolation  of  B.  coli. 

No  merely  presumptive  test  of  this  sort  should  be  sub- 
stituted for  the  complete  demonstration  of  B.  coli  in  the 
detailed  sanitary  study  of  a  special  source  of  supply.  For 
extensive  routine  surveys  of  considerable  series  of  samp- 
ling stations,  on  the  other  hand,  the  bile  test  offers  a  satis- 
factory approximation  to  the  truth. 

The  litmus-lactose-agar  plate  furnishes  a  presumptive 
test  of  considerable  value  as  indicated  in  Chapter  IV, 
although  it  is  probably  less  delicate  than  the  fermentation 
methods.  With  polluted  waters,  however,  comparative 
studies  of  the  agar  plate  and  the  bile  method  are  much  to 
be  desired. 

Other  special  media  have  been  suggested  for  rapid 
routine  water  analysis  of  which  those  containing  "  neutral 
red,"  one  of  the  safranine  dyes,  have  been  most  fully  studied. 
Rothberger  (Rothberger,  1898)  first  pointed  out  that  B. 
coli  reduces  solutions  of  this  substance,  the  color  chang- 
ing to  canary-yellow  accompanied  by  green  fluorescence. 
Makgill  (Makgill,  1901),  Savage  (Savage,  1901),  and 
other  English  observers,  as  well  as  Braun  (1906),  in 
France,  report  favorable  results  from  the  use  of  this  test, 


Presumptive   Tests  for  B.   Coli.  151 

but  according  to  American  standards,  Irons  (Irons,  1902) 
and  Gage  and  Phelps  (Gage  and  Phelps,  1903)  conclude 
that  the  group  of  organisms  giving  a  positive  neutral  red 
reaction  is  too  large  a  one  to  give  very  valuable  sanitary 
information. 

Stokes  (1904)  urged  the  use  of  lactose  broth  with  the 
addition  of  neutral  red,  and  believed  that  the  production 

2 

in  this  medium  of  30-50  per  cent  of  gas  with  a  —  gas  for- 
mula and  the  change  of  neutral  red  to  canary  yellow  in  the 
closed  arm  of  the  fermentation  was  characteristic  for  B. 
coli. 

The  lactose-bile  method,  however,  is  the  only  rapid 
test  whose  value  has  yet  been  established  by  a  considerable 
series  of  investigations;  it  seems  to  be  the  best  presumptive 
test  now  available. 


CHAPTER  IX. 

OTHER  INTESTINAL   BACTERIA. 

IT  would  be  an  obvious  advantage  if  the  evidence  of 
sewage  contamination,  furnished  by  the  presence  of  B. 
coli,  could  be  reinforced  and  confirmed  by  the  discovery 
in  water  of  other  forms  equally  characteristic  of  the 
intestinal  canal.  The  attention  of  bacteriologists  in 
England  and  America  has  been  turned  in  this  direction 
during  the  past  few  years;  and  two  groups  of  organisms, 
the  sewage  streptococci  and  the  anaerobic  spore-bearing 
bacilli,  have  been  described  as  probably  significant. 

The  term  "sewage  streptococci,"  as  generally  used, 
covers  an  ill-defined  group  including  many  cocci  which 
do  not  occur  in  well-marked  chains.  Those  most  com- 
monly found  correspond  rather  closely  to  the  type  of 
Str.  pyogenes  (identical  with  Str.  erysipelatos).  They 
grow  feebly  on  the  surface  of  ordinary  nutrient  agar,  pro- 
ducing faint  transparent,  rounded  colonies,  but  under 
semi-anaerobic  conditions  flourish  better,  giving  a  well- 
marked  growth  along  the  gelatin  stab  and  only  a  small 
circumscribed  film  on  the  surface.  They  are  favored  by 
the  presence  of  the  sugars  and  ferment  dextrose  and 
lactose,  with  the  formation  of  abundant  acid  but  no  gas. 


Other  Intestinal  Bacteria.  153 

They  are  seen  under  the  microscope  as  cocci,  occurring 
as  a  rule  in  pairs,  short  chains,  or  irregular  groups.  They 
do  not  show  visible  growth  and  do  not  form  indol  and 
nitrite  in  the  standard  peptone  and  nitrate  solutions;  most 
of  them  do  not  liquefy  gelatin,  though  occasionally  forms 
are  found  which  possess  this  power,  Until  recently,  no 
systematic  study  of  the  various  species  found  in  the  intes- 
tine had  been  made  and  at  present  all  cocci  giving  the 
characteristic  growth  on  agar  and  strongly  fermenting 
lactose  are  commonly  included  as  "sewage  streptococci.1 ' 
Although  the  significance  of  the  streptococci  as  sewage 
organisms  is  not  established  with  the  same  definiteness 
which  marks  our  knowledge  of  the  colon  group,  these 
forms  have  been  isolated  so  frequently  from  polluted 
sources  and  so  rarely  from  normal  ones  that  it  now  seems 
reasonable  to  regard  their  presence  as  indicative  of  pollu- 
tion. Although  originally  reported  by  Laws  and  Andrewes 
(Laws  and  Andrewes,  1894),  their  importance  was  not 
emphasized  until  1899  and  1900,  when  Houston  (Houston, 
i899b,  i9cob)  laid  special  stress  upon  the  fact  that  strep- 
tococci and  staphylococci  seem  to  be  characteristic  of 
sewage  and  animal  waste,  the  former  being,  in  his  opinion, 
the  more  truly  indicative  of  dangerous  pollution,  since 
they  are  "readily  demonstrable  in  waters  recently  polluted 
and  seemingly  altogether  absent  from  waters  above  sus- 
picion of  contamination."  In  six  rivers,  recently  exten- 
sively sewage-polluted,  he  found  streptococci  in  from  one- 
tenth  to  one  ten-thousandth  of  a  c.c.of  the  water  examined, 


154  Elements  of  Water  Bacteriology. 

although  in  some  cases  the  chemical  analysis  would  not 
have  indicated  dangerous  pollution.  On  the  other  hand,- 
eight  rivers,  not  extensively  polluted,  showed  no  strep- 
tococci in  one-tenth  of  a  c.c.,  although  the  chemical  and 
the  ordinary  bacteriological  tests  gave  results  which  would 
condemn  the  waters.  Horrocks  (Horrocks,  1901)  found 
these  organisms  in  great  abundance  in  sewage  and  in 
waters  which  were  known  to  be  sewage-polluted,  but 
which  contained  no  traces  of  Bacillus  coli.  He  found  by 
experiment  that  B.  coli  gradually  disappeared  from  speci- 
mens of  sewage  kept  in  the  dark  at  the  temperature  of  an 
outside  veranda,  while  the  commonest  forms  which  per- 
sisted were  varieties  of  streptococci  and  staphylococci. 

In  America  attention  was  first  called  to  these  organisms 
by  Hunnewell  and  one  of  us  (Winslow  and  Hunnewell, 
1902*),  and  the  same  authors  later  (Winslow  and  Hunne- 
well, i902b)  recorded  the  isolation  of  streptococci  from  25 
out  of  50  samples  of  polluted  waters.  Gage  (Gage,  1902), 
from  the  Lawrence  Experiment  Station,  has  reported 
the  organisms  present  in  the  sewage  of  that  city,  while 
Prescott  (i902b)  has  shown  that  they  are  abundant  in 
fecal  matter  and  often  overgrow  B.  coli  in  a  few  hours 
when  inoculations  are  made  from  such  material  into 
dextrose  broth.  In  the  recent  monograph  of  Le  Gros 
(Le  Gros,  1902)  of  the  many  streptococci  described,  all 
without  exception  were  isolated,  either  from  the  body  or 
from  sewage.  Baker  and  one  of  us  (Prescott  and  Baker, 
1904)  found  these  organisms  present  in  each  of  50  samples 


Other  Intestinal  Bacteria.  155 

of  polluted  wafers.  On  the  other  hand,  in  the  study  of 
259  samples  of  presumably  unpolluted  waters,  by  the 
method  of  direct  plating,  Nibecker  and  one  of  the  authors 
(Winslow  and  Nibecker,  1903)  found  streptococci  in  only 
one  sample.  Gordon  (1904)  showed  that  certain  strep- 
tococci are  abundant  in  normal  saliva  and  are  found 
in  air  which  has  been  exposed  to  human  pollution  but  not 
in  normal  air.  On  the  whole  there  can  be  no  doubt  of 
the  fact  that  streptococci  occur  on  the  surfaces  of  the 
human  and  animal  body  more  commonly  than  anywhere 
else  in  nature. 

The  isolation  of  these  organisms  either  from  plates  or 
liquid  cultures  is  easy.  On  the  lactose-agar  plate,  made 
directly  from  a  polluted  water,  the  colonies  of  the  strep- 
tococci may  generally  be  distinguished  from  those  of  other 
acid-formers  by  their  small  size,  compact  structure,  and 
deep-red  color,  which  is  permanent,  never  changing  to 
blue  at  a  later  period  of  incubation.  Developing  some- 
what slowly,  however,  they  may  be  overlooked  if  present 
only  in  small  numbers.  In  the  dextrose-broth  tube,  strep- 
tococci will  generally  appear  in  abundance  after  a  suit- 
able period  of  incubation.  Prescott  and  Baker,  in  the 
work  above  mentioned,  found  that  with  mixtures  of  B. 
coli  and  streptococci  in  which  the  initial  ratios  of  the 
latter  to  the  former  varied  from  i:  94  to  208:  i,  the  colon 
bacilli  developed  rapidly  during  the  early  part  of  the 
experiment,  reaching  a  maximum  after  about  fourteen 
hours,  and  then  diminishing  rapidly.  The  streptococci 


156 


Elements  of  Water  Bacteriology. 


first  became  apparent  after  ten  to  fifteen  hours  and 
reached  their  maximum  after  twenty  to  sixty  hours,  accord- 
ing to  the  number  originally  present. 

Applying  the  same  method  to  polluted  waters,  similar 
periodic  changes  were  observed;  pure  cultures  of  B.  coli 
were  first  obtained,  then  the  gradual  displacement  of  one 
form  by  the  other  took  place,  and  at  length  the  streptococci 
were  present  either  in  pure  culture  or  in  great  predomi- 
nance as  shown  by  the  accompanying  tables.  The  samples 
of  water  were  plated  directly  upon  litmus  lactose  agar  and 
the  plates  were  incubated  at  37°  for  twenty-four  hours, 


TABLE  I. 

RELATIVE  GROWTH   OF  B.  COLI  AND  SEWAGE  STREPTOCOCCI 
FROM  POLLUTED  WATERS  IN  DEXTROSE  BROTH. 
(PRESCOTT  AND   BAKER,   1904.) 


i 
4 

2 

10 

3 
9 

4 

5 
8 

6 

55 
400 

0 

7 
35 

8 
460 

9 

10 

105 
410 

0 

410 

Red  colonies  developing  from  i  ) 
c.c.  of  original  sample  on  litmus   ? 

5 
200 

0 

270 

1250 
420 

0 

285 

Number  found,   in 
millions  per  cubic 
centimeter,    after 
growth  in  dextrose  " 
broth  for  various 
periods  .... 

ii 
hrs. 

16 
hrs. 

B.  coli 
Strept. 

o 

0 
200 

20 
0 

68 

0 

185 
o 

130 

0 

332 

0 

B.  coli 

76 

13° 

220 

2IO 

140 

420 

Strept. 

40 

25 

20 

IO 

45 

30 

20 

210 

75 

145 

hrl 
h3?, 

B.  coli 

280 

ISO 

385 

370 

300 

570 

200 

405 

320 

300 

350 

Strept. 

140 

85 
0 

420 

280 
25 
^80" 

170 

300 

0 

300 

I7OO 
210 
170 

no 

20 

350 
24 
105 

37° 
105 
250 

B.  coli 

O 

474 

no 

300 

Strept. 

400 

& 

hrs. 

B.  coli 

0 

o 

O 

0 

o 

I 

12 

2 

8 

45 

10 

O 

ISO 

o 
86 

o 

170 

Strept. 

2 

0 

0 

45 

First  gas  noted  after  (hrs.). 

10 

10 

9 

9 

10 

8 

6 

6 

8 

Other  Intestinal  Bacteria. 


157 


when  the  red  colonies  were  counted.  At  the  time  of 
plating,  i  c.c.  fro'm  each  sample  was  also  inoculated  into 
dextrose  broth  in  fermentation  tubes,  which  were  likewise 
incubated  at  37°.  After  various  periods,  as  indicated 
by  the  table  below,  the  tubes  were  shaken  thoroughly 
and  i  c.c.  of  the  contents  withdrawn.  This  was  diluted 
(generally  1-1,000,000)  with  sterile  water,  plated  on  litmus 


TABLE   II. 

RELATIVE  GROWTH  OF  B.  COLI  AND  SEWAGE  STREPTOCOCCI 
FROM  POLLUTED    WATERS  IN  DEXTROSE  BROTH. 

(PRESCOTT  AND  BAKER,  1904.) 


18 

19 

20 

21 

22 

23 

24 

25 

Red  colonies  developing  from  i  c.c.  of  origi-  ) 
nal  sample  on  litmus  lactose  agar    .    .    .    J 

i 

150 

25 

30 

So 

170 

200 

30 

Number  found,  in  millions 
per  cubic  centimeter,  after 
growth  in  dextrose  broth  ' 
for  various  periods  .    .    - 

his. 

B.  coli 

.02 

.    . 

.01 

.04 

.12 

•55 

1.6 

Strept. 

0 

0 

o 

O 

o 

0 

h£ 

B.  coli 

266 

IOO 

88 

350 

510 

380 

330 

1  60 

Strept. 

150 

o 
610 

40 
72 

140 

240 

128 

80 

22O 

£. 
h£. 

B.  coli 

520 

700 

IOOO 

740 

IOO 

300 
3000 

27 

30 

Strept. 

800 

0 

252 

10 

40 

860 

670 

1080 

22 
22 

2O 

2500 

36 

66 

70 

4380 

7 
60 
35 

7 
52 

10 

B.  coli 

0 

10 

260 
38 
3-8 

Strept. 

330 

16 
16 

£. 

B.  coli 

Strept. 

31 

4i 

25 

IO 

lactose  agar  in  the  usual  way,  and  incubated  for  twenty- 
four  hours.  The  colonies  of  B.  coli  and  streptococci 
were  distinguished  microscopically,  and  by  difference  in 
color  and  general  characters. 


158  Elements  of  Water  Bacteriology. 

The  successive  growth  of  these  two  intestinal  groups  in 
the  same  dextrose-broth  tube  suggests  the  following  method 
for  the  detection  of  both  B.  coli  and  sewage  streptococci: 

Inoculate  the  desired  quantity  of  water,  preferably  i  c.c., 
into  'dextrose  broth,  in  a  fermentation  tube,  and  incubate 
at  37  degrees.  After  a  few  hours'  incubation  examine 
the  cultures  for  gas.  Within  two  or  three  hours  after  gas 
formation  is  first  evident,  plate  from  the  broth  in  litmus 
lactose  agar,  incubating  for  twelve  to  eighteen  hours  at 
37  degrees.  If  at  the  end  of  this  time  no  acid-producing 
colonies  are  found,  it  is  probably  safe  to  assume  that 
there  were  no  colon  bacilli  present.  On  the  other 
hand,  if  red  colonies  develop,  these  must  be  further 
examined  by  the  regular  diagnostic  tests  for  B.  coli. 
After  the  first  plating  from  the  dextrose  broth,  replace 
the  fermentation  tube  in  the  incubator  and  allow  it  to 
remain  for  twenty-four  to  thirty-six  hours,  then  plate 
again  in  litmus  lactose  agar.  This  plating  should  give  a 
nearly  pure  culture  of  streptococci  if  these  organisms  were 
originally  present  in  the  water. 

The  relative  relation  of  the  streptococci  and  the  colon 
bacilli  to  sewage  pollution  is  still  somewhat  uncertain. 
Houston  (Houston,  1900)  held  that  the  former  microbes 
imply  "animal  pollution  of  extremely  recent  and  therefore 
specially  dangerous  kind."  Horrocks  (Horrocks,  1901), 
on  the  other  hand,  maintains,  largely  on  the  strength  of 
certain  experiments  with  stored  sewage,  that  the  strep- 
tococci persist  after  colon  bacilli  have  disappeared  and 


Other  Intestinal  Bacteria.  159 

indicate  contamination  with  old  sewage  which  is  not 
necessarily  dangerous.  These  discordant  results  are  prob- 
ably to  be  explained  by  the  different  media  in  which 
the  viability  of  the  bacteria  was  compared.  It  seems 
likely  that  in  sewage  where  there  is  a  large  amount  of 
organic  food  material  present  the  streptococci  may  kill 
out  the  colon  bacilli  as  they  do  in  the  fermentation  tube. 
This  would  explain  Hbrrocks'  results.  On  the  other 
hand,  there  is  good  evidence  that  the  streptococci  are  less 
resistant  than  B.  coli  to  the  unfavorable  conditions  which 
exist  in  water  of  ordinary  organic  purity.  In  waters  of 
potable  character  B.  coli  is  frequently  present  without  the 
streptococcus;  and  a  negative  test  for  streptococci  has 
little  significance.  A  positive  test  on  the  other  hand  fur- 
nishes valuable  confirmatory  evidence  of  pollution.  This 
evidence  is  of  course  of  special  importance  when,  through 
the  activity  of  the  streptococci  themselves,  or  from  any 
other  cause,  the  colon  isolation  has  yielded  an  erroneous 
negative  result. 

The  English  Committee  appointed  to  consider  the 
standardization  of  methods  for  the  bacterioscopic  exami- 
nation of  water  (1904),  by  a  majority  vote  recommended 
the  enumeration  of  streptococci  as  a  routine  procedure  in 
sanitary  water  analysis,  and  the  test  deserves  more  careful 
attention  than  it  has  yet  received  in  America. 

There  seems  even  reason  to  hope  that  the  streptococci 
may  prove  of  assistance  in  the  important  task  of  differ- 
entiating human  and  animal  pollution,  a  task  in  which 


160  Elements  of  Water  Bacteriology. 

all  other  tests  have  so  far  failed.  Unlike  the  colon  bacilli, 
streptococci  from  the  intestines  of  cattle  and  men  appear 
to  belong  to  distinct  types.  The  recognition  of  this  fact 
we  owe  primarily  to  Gordon  (1905),  who  made  an  elabo- 
rate study  of  the  fermentative  power  of  the  streptococci  in 
a  long  series  of  carbohydrate  media.  His  work  and  that 
of  Houston  (Houston,  1904;  Houston,  1905*;  Houston, 
i9<D5b)  have  made  it  clear  that  the  streptococci  of  the 
herbivora  differ  from  those  found  in  the  human  body  in 
their  low  fermentative  power.  In  a  recent  review  of  the 
genus,  Andre wes  and  Horder  (1906)  describe  the  type 
characteristic  of  the  herbivora  under  the  name,  Str. 
equinus,  and  define  it  by  its  failure  to  ferment  lactose, 
ramnose,  inulin  or  mannite,  or  to  reduce  neutral  red. 
Five  other  types  are  described  from  the  human  mouth 
and  intestine;  all  of  them  ferment  lactose,  and  most  reduce 
neutral  red  and  ferment  ramnose.  The  commonest  intes- 
tinal form  clots  milk,  reduces  neutral  red  and  ferments 
saccharose,  salicin,  coniferin,  and  mannite.  The  specific 
types  of  the  genus  streptococcus,  grade  into  each  other  by 
almost  imperceptible  degrees,  and  streptococci  fermenting 
lactose  and  raffinose  and  reducing  neutral  red  are  some- 
times found  in  bovine  feces.  The  fact  that  Str.  equinus 
is  the  commonest  form  among  herbivora  and  a  rarer  form 
in  man  will  however  prove  an  important  contribution  to 
water  bacteriology,  if  confirmed  by  other  observers. 

The  English  bacteriologists  have  ascribed  much  impor- 
tance as  indicators  of  sewage  pollution  to  another  group 


Other  Intestinal  Bacteria.  161 

of  organisms,  the  anaerobic  spore-forming  bacilli,  of  which 
the  B.  sporogenes  is  a  type.  This  form  was  isolated  by 
Klein  (Klein,  1898;  Klein,  1899)  in  1895,  in  the  course 
of  an  epidemic  of  diarrhoea  at  St.  Bartholomew's  Hos- 
pital, and  described  under  the  name  of  B.  enteritidis- 
sporogenes;  it  is  closely  related  to  the  B.  aerogenes  capsu- 
latus  of  Welch  (Welch  and  Nuttall,  1892). 

Klein's  procedure  for  isolating  the  B.  sporogenes  is 
simple  in  the  case  of  highly  polluted  waters.  A  portion 
of  the  sample  to  be  examined  is  added  to  a  tube  of  sterile 
milk,  which  is  then  heated  to  80°  C.  for  ten  minutes 
to  destroy  vegetative  cells.  The  milk  is  next  cooled  and 
incubated  under  anaerobic  conditions,  which  may  be 
accomplished  most  conveniently  by  Wright's  method.  A 
tight  plug  of  cotton  is  forced  a  quarter  way  c\own  the  test 
tube,  the  space  above  is  loosely  filled  with  pyrogallic  acid, 
a  few  drops  of  a  strong  solution  of  caustic  potash  are 
added,  and  the  tube  is  tightly  closed  with  a  rubber  stopper. 
After  eighteen  to  thirty-six  hours  at  37  degrees  the  appear- 
ance of  the  tube  will  be  characteristic  if  the  B.  sporogenes 
is  present.  "The  cream  is  torn  or  altogether  dissociated 
by  the  development  of  gas,  so  that  the  surface  of  the 
medium  is  covered  with  stringy,  pinkish-white  masses  of 
coagulated  casein,  enclosing  a  number  of  gas-bubbles. 
The  main  portion  of  the  tube  formerly  occupied  by  the 
milk  now  contains  a  colorless,  thin,  watery  whey,  with  a 
few  casein  lumps  adhering  here  and  there  to  the  sides  of 
the  tube.  When  the  tube  is  opened,  the  whey  has  a  smell 


1 62  Elements  of  Water  Bacteriology. 

of  butyric  acid  and  is  acid  in  reaction.  Under  the  micro- 
scope the  whey  is  found  to  contain  numerous  rods,  some 
motile,  others  motionless." 

The  B.  sporogenes  when  isolated  in  pure  culture  on 
glucose  agar  is  a  stout  rod.  It  liquefies  gelatin,  forming 
in  this  medium  large  oval  spores.  It  is  strongly  patho- 
genic for  guinea  pigs,  by  which  character  it  is  distinguished 
from  the  B.  butyricus  of  Botkin. 

The  researches  of  Klein  and  Houston  (Klein  and 
Houston,  1898,  1899)  have  shown  that  the  B.  sporogenes 
occurs  in  English  sewage  in  numbers  varying  from  30  to 
2200  per  c.c.  and  that  it  is  often  absent  in  considerable 
volumes  of  pure  water.  In  Boston  sewage  it  may  usually 
be  isolated  from  .01  or  .001  of  a  c.c.  (Winslow  and 
Belcher,  1904). 

Since  this  organism  is  not  present  in  very  large  num- 
bers, even  in  sewage,  the  test  of  a  water  supply  must  be 
made  with  large  samples,  and  the  concentration  of  at  least 
2000  c.c.  of  water  by  filtration  through  a  Pasteur  filter 
is  recommended  by  Horrocks  as  a  necessary  prelude 
(Horrocks,  1901).  Since  the  spores  of  an  anaerobic 
bacillus  may  persist  for  an  indefinite  period  in  polluted 
waters,  their  presence  need  not  necessarily  indicate 
recent  or  dangerous  pollution.  On  the  whole,  it  does 
not  appear  that  the  practical  application  of  the  anaerobic 
test  will  ever  be  a  wide  one. 

Besides  the  streptococci  and  the  anaerobic  spore  formers, 
various  intestinal  bacilli,  more  or  less  closely  allied  to  the 


Other  Intestinal  Bacteria. 


B.  coli  group,  have  received  attention  from  the  English 
bacteriologists.  A  large  number  of  species  and  races 
have  been  described  which  are  intermediate  in  their 
properties  between  B.  typhi  and  B.  coli,  all  being  non- 
spore-forming,  non-liquefying  rods,  which  produce  a 
more  or  less  characteristic  growth  on  solid  media. 
Durham  (1898)  divided  these  forms  into  three  main 
divisions,  grouped,  respectively,  about  B.  typhi,  B.  enteri- 
tidis  and  B.  coli.  Organisms  of  the  first  division  ferment 
neither  dextrose,  lactose  nor  saccharose;  those  of  the 
second  ferment  dextrose  but  not  lactose;  and  those  of 
the  B,  coli  division  form  gas  in  both  these  sugars.  The 
relationship  of  the  commonest  species  is  indicated  in 
tabular  form  below: 

BACTERIA  OF  THE  COLON-TYPHOID  GROUP. 


Species. 

Dextrose. 

Lactose. 

Gas  For- 
mation. 

Acid  Pro- 
duction. 

Gas  For- 
mation. 

Acid  Pro- 
duction. 

B.  alcaligenes  

None 
None 
None 

Active 
Active 
Active 

Active 

None 
Slight 
Distinct 

Strong 
Strong 
Strong 

Strong 

None 
None 
None 

None 
None 
None 

Active 

None 
Slight 
Slight 

Slight 
Slight 
Slight 

Strong 

B.  typhi 

B.  dysenteriae  

B.  enteritidis  
Paratyphoid  bacilli  .... 
Hog  cholera  bacillus  .  .  . 

B.  coli  

In  the  typhoid  division,  B.  alcaligenes  and  B.  dysen- 
teriae are  the  best  known '  forms,  besides  B.  typhi  itself. 


164  Elements  of  Water  Bacteriology. 

B.  alcaligenes  stands  at  the  lower  end  of  the  whole  series 
in  fermentative  power.  B.  typhi  forms  a  slight  initial 
acidity  in  milk  and  a  slight  acidity  in  dextrose  broth, 
while  the  reaction  of  B.  alcaligenes  in  sugar  media  is 
always  alkaline.  B.  dysenteriae,  on  the  other  hand, 
differs  from  B.  typhi  in  the  direction  of  the  B.  enteritidis 
group,  producing  a  well-marked  acid  reaction,  but  no  gas 
in  dextrose  media.  B.  typhi  and  B.  dysenteriae  are,  of 
course,  also  distinguished  by  their  specific  serum  reactions. 
Neither  B.  alcaligenes  nor  any  other  member  of  this 
group  (except  the  disease-producers  themselves,  when 
clearly  identified  by  agglutination  tests)  has  any  well- 
established  sanitary  significance.  Non-acid-forming  bac- 
teria of  this  general  type  are  frequently  found  in  feces, 
but  they  are  also  found  in  other  habitats,  and  comparative 
data  are  entirely  lacking  to  show  that  they  are  more 
abundant,  proportionately,  in  polluted  than  in  normal 
waters. 

The  second  great  division  of  the  colon-typhoid  bacteria 
is  the  hog  cholera  group,  or  the  Gartner  group,  as 
Durham  (1898)  called  it.  As  defined  by  him,  it  differed 
from  the  typhoid  group  by  gas  formation  in  dextrose, 
and  from  the  colon  group  by  the  production  of  a  final 
alkaline  reaction  in  milk.  It  includes  the  Gartner  bacillus 
(B.  enteritidis),  the  hog  cholera  bacillus  (B.  cholerae 
suis),  and  the  paratyphoid  bacilli.  Some  of  these  forms, 
the  paratyphoid  bacilli,  for  example,  and  B.  enteritidis 
(isolated  in  cases  of  meat  poisoning),  produce  intestinal 


Other  Intestinal  Bacteria.  165 

disease  in  man.  Unless,  however,  specific  pathogenes 
can  be  identified  by  serum  reactions,  the  non-lactose- 
fermenting  forms  have  no  clear  significance  in  water 
analysis.  They  occur  in  the  intestine,  though  not  appar- 
ently in  great  abundance.  Houston  (1904)  attempted 
to  isolate  bacteria  fermenting  dextrose  but  not  lactose 
from  normal  human  stools;  but  out  of  257  colonies  studied 
only  six  failed  to  form  acid  and  gas  in  lactose  media. 
Organisms  of  this  character  may  also  be  found  in  unpol- 
luted water,  as  shown  by  the  occurrence  of  positive 
dextrose-broth  tests  followed  by  negative  litmus-lactose- 
agar  plates  (see  Chapter  VIII). 

Organisms  belonging  to  the  colon  group  itself,  and 
producing  gas  and  acid  in  both  dextrose  and  lactose 
media,  are  much  more  clearly  related  to  sewage  pollution. 
Numerous  investigations  of  normal  waters  have  shown 
that  bacteria  possessing  these  two  properties  are  rarely 
found  where  pollution  is  absent,  while  the  commonest 
intestinal  forms  exhibit  this  dual  fermentative  power. 
The  mass  of  evidence  establishing  excretal  -origin  is 
stronger  in  the  case  of  B.  coli  than  for  any  of  its  allies. 
B.  communior,  which  differs  in  possessing  the  power  of 
fermenting  saccharose,  is,  however,  almost  invariably 
included  with  B.  coli  in  practical  work.  The  signifi- 
cance of  those  forms  which  differ  from  B.  coli  by  lacking 
one  or  all  of  the  properties  of  motility,  of  indol  formation, 
or  of  nitrate  reduction,  or  by  failing  to  coagulate  milk  in 
the  standard  time,  is  somewhat  less  clear.  B.  aerogenes 


1 66  Elements  of  Water  Bacteriology. 

is  a  well-defined  species  of  this  group,  which  differs  from 
B.  coli  in  lacking  motility,  in  possessing  a  capsule,  in 
forming  a  somewhat  heavier  growth  on  media  than  B. 
coli,  and  in  failing  to  form  indol.  This  and  other  allied 
forms  are  sometimes  called  "atypical  B.  coli,"  or  "para- 
colon  bacilli,"  and  Vincent  gives  them  the  picturesque 
name,  "microbic  satellites  of  B.  coli." 

Sometimes,  as  pointed  out  in  Chapter  VI,  these  organ- 
isms are  merely  weakened  strains  of  B.  coli  which  have 
lost  certain  powers  through  exposure  to  unfavorable  envi- 
ronment. The  results  obtained  by  Peckham  (1897)  suggest 
that  the  indol  reaction  in  particular  is  highly  variable. 
By  successive  daily  transfers  in  peptone  broth  she  was 
able  to  increase  the  amount  of  indol  produced  by  normal 
B.  coli,  and  by  a  longer  continuance  of  the  same  process 
to  again  weaken  and  abolish  the  power  of  forming  it. 
Gas  formation  too  was  slackened  in  the  cultures  grown 
for  too  many  transfers  in  the  same  medium.  Horrocks 
(1903)  found  that  B.  coli  kept  in  unsterilized  well  waters 
and  tap  Waters  and  in  sterilized  sewage  and  Thames 
water  for  two  to  three  months,  showed  only  a  feeble  indol 
production  and  a  delayed  action  on  milk  and  neutral 
red.  Even  the  fermentative  powers  which  distinguish 
the  B  coli  and  the  B.  enteritidis  groups  may  be  modified 
by  environmental  conditions.  Twort  (1907)  reports 
that  by  continued  cultivation  in  sugar  media  he  was 
able  to  develop  fermentative  power  in  certain  members 
of  the  Gartner  group  which  lacked  such  powers  before. 


Other  Intestinal  Bacteria.  167 

It  is  certain  that  in  the  intestine  these  atypical  forms 
are  less  common  than  the  colon  bacillus  itself,  bearing  to 
it  indeed  very  much  the  relation  implied  in  Vincent's  term, 
satellites.  Houston  (i903a)  examined  in  detail  101 
cultures  of  coli-like  microbes  isolated  from  feces,  and 
found  that  72  per  cent  of  the  cultures  were  typical  in  all 
respects,  while  n  per  cent  more  differed  only  in  being 
non-motile.  The  remaining  17  per  cent  were  atypical, 
reacting  abnormally  to  milk,  indol,  neutral  red,  litmus 
whey  or  Capaldi  and  Proskauer's  medium.  In  a  later 
investigation,  Houston  (1904)  made  a  careful  study  of 
the  distribution  of  the  atypical  forms  in  feces,  sewage, 
polluted  water,  and  the  filtered  water-supplies  of  London. 
According  to  his  ingenious  system  of  nomenclature, 
"fl"  indicates  an  organism  which  produces  green 
fluorescence  in  neutral  red  broth;  "ag,"  one  which  forms 
acid  and  gas  in  lactose  media;  "in,"  one  which  produces 
indol;  and  "ac,"  one  which  acidifies  and  clots  litmus 
milk.  The  combination  of  all  these  properties  gives 
"Flaginac,"  or  typical  B.  coli;  "aginac,"  is  a  form  which 
fails  to  reduce  neutral  red;  "flagac,"  one  which  fails  to 
form  indol,  etc.  "Flaginac"  B.  coli  form  the  great 
majority  of  coli-like  microbes  in  feces,  but  Houston  found 
that  in  filtered  water  they  are  outnumbered  by  atypical 
forms,  of  which  he  recognized  thirty-five  distinct  types. 

On  the  whole,  all  the  evidence  tends  to  the  assumption 
that  the  atypical  forms,  or  "paracolon  bacilli,"  generally 
represent  weakened  strains  from  the  intestinal  B.  coli 


1 68  Elements  of  Water  Bacteriology. 

stock.  As  Savage  says,  "  we  know  that  nearly  all  the  coli- 
like  organisms  in  feces  are  quite  typical  B.  coli,  that  in- 
sewage  a  good  many  atypical  varieties  are  present,  and 
that  in  contaminated  water  and  soil  the  proportion  present 
is  still  larger."  The  presence  of  these  forms  in  water 
must,  in  the  light  of  present  knowledge,  be  considered 
suspicious,  though  not  an  indisputable  evidence  of  con- 
tamination. The  only  safe  rule,  when  atypical  forms  are 
found  without  standard  B.  coli,  is  to  secure  another  sample 
for  examination.  Fortunately  this  condition  rarely  occurs, 
since  typical  colon  bacilli  are  generally  found  in  duplicate 
samples  when  the  atypical  forms  are  present. 

B.  cloacae,  which  differs  from  B.  coli  in  the  liquefaction 
of  gelatin,  stands  in  much  the  same  position  as  the  atypical 
forms  of  the  colon  bacillus.  We  have  seen  that  organisms 
which  ferment  dextrose  and  lactose  are  rarely  found  in 
normal  waters,  and  this  form  must,  therefore,  be  regarded 
with  suspicion.  Detailed  studies  of  its  distribution  are 
necessary,  however,  before  its  presence  can  be  given  the 
same  weight  as  that  of  the  colon  bacillus. 

There  are  numerous  other  sewage  bacteria  whose 
presence  is  more  or  less  characteristic  of  polluted  waters. 
Organisms  of  the  Proteus  group  are  sometimes  present, 
exhibiting  marked  morphological  variations,  from  the 
coccus  form  to  long  twisted  threads,  and  forming  on 
gelatin  irregular  amoeboid  colonies  with  filiform  processes 
extending  into  the  surrounding  gelatin.  The  B.  subtilis 
group  of  strongly  aerobic  spore-forming  bacilli,  giving  a 


OtJier  Intestinal  Bacteria.  169 

brown  wrinkled  parchment-like  growth  on  agar,  and 
moss-like  liquefying  colonies  on  gelatin,  is  usually  repre- 
sented; but  this  form  is  more  characteristic  of  decaying 
vegetation  in  the  surface  layers  of  the  soil  than  it  is  of 
sewage.  Vincent  (1907)  and  other  French  observers 
consider  the  determination  of  the  total  number  of  anaerobic 
bacteria  as  significant  since  the  decomposition  of  organic 
matter  is  accompanied  by  anaerobic  growth.  It  is  not 
claimed,  however,  that  bacteria  of  this  type  are  character- 
istic of  animal  more  than  of  vegetable  decompositions,  and 
the  total  anaerobic  count  apparently  adds  nothing  of  im- 
portance to  the  information  gained  by  the  ordinary  gelatin 
plate  method.  The  property  of  liquefaction  was  formerly 
believed  to  be  of  significance,  inasmuch  as  the  liquefying 
bacteria  were  regarded  as  indicative  of  pollution.  This 
position  is,  however,  no  longer  tenable,  since  many  bacteria, 
typical  of  the  purest  waters,  may  cause  liquefaction. 

As  Savage  says  in  summing  up  this  question:  "The 
number  of  different  species  of  organisms  in  sewage  is  very 
great,  and  it  is  highly  probable  that  many  of  them  occur 
in  all  specimens  of  ordinary  sewage;  but,  except  for  B. 
coli,  streptocci,  and  B.  enteritidis  sporogenes,  their  presence 
has  not  been  ascertained  with  sufficient  constancy,  nor  has 
their  numerical  occurrence  been  sufficiently  investigated 
to  make  them  of  value  as  indicators  of  sewage  pollution.'* 
(Savage,  1906.) 


CHAPTER  X. 

THE    SIGNIFICANCE   AND   APPLICABILITY   OF    THE 
BACTERIOLOGICAL   EXAMINATION. 

THE  first  attempt  of  the  expert  called  in  to  pronounce 
upon  the  character  of  a  potable  water  should  be  to  make 
a  thorough  sanitary  inspection  of  the  pond,  stream,  well, 
or  spring  from  which  it  is  derived.  Study  of  the  possible 
sources  of  pollution  on  a  watershed,  of  the  direction  and 
velocity  of  currents  above  and  below  ground,  of  the  char- 
acter of  soil  and  the  liability  to  contamination  by  sur- 
face-wash are  conceded  to  yield  evidence  of  the  greatest 
value.  Often,  however,  an  opinion  is  desired  as  to  the 
quality  of  water  sent  from  a  distance  without  the  oppor- 
tunity of  examining  its  surroundings;  and  even  when 
sanitary  inspection  can  be  made,  its  results  are  by  no 
means  conclusive.  If  house  or  barnyard  drainage  or 
sewage  is  actually  seen  to  enter  a  water  used  for  drinking 
purposes  it  is  obviously  unnecessary  to  carry  out  delicate 
chemical  or  bacteriological  tests  to  detect  pollution.  On 
the  other  hand,  no  reconnoissance  can  show  certainly 
whether  unpurified  drainage  from  a  cesspool  does  or  does 
not  reach  a  given  well;  whether  sewage  discharged  into 
a  lake  does  or  does  not  find  its  way  to  a  neighboring 

170 


Bacteriological  Examination.  171 

intake;  whether  pollution  of  a  stream  has  or  has  not 
been  removed  by  a  certain  period  of  flow.  Evidence 
upon  these  points  must  be  obtained  from  a  careful  study 
of  the  characteristics  of  the  water  in  question,  and  this 
study  can  be  carried  out  along  two  lines,  chemical  and 
bacteriological. 

A  chemical  examination  of  water  for  sanitary  purposes 
is  mainly  useful  in  throwing  light  upon  one  point  —  the 
amount  of  decomposing  organic  matter  present.  It  also 
gives  an  historical  picture  which  may  be  of  some  value 
or  suggestiveness.  Humus-like  substances  may  be  abun- 
dant in  surface-waters  quite  free  from  harmful  pollution, 
but  these  are  stable  compounds.  Easily  decomposable 
bodies,  on  the  other  hand,  must  obviously  have  been 
recently  introduced  into  the  water  and  mark  a  transi- 
tional state.  "The  state  of  change  is  the  state  of  danger," 
as  Dr.  T.  M.  Drown  once  phrased  it.  Sometimes  the 
organic  matter  has  been  washed  in  by  rain  from  the  sur- 
face of  the  ground,  sometimes  it  has  been  introduced  in 
the  more  concentrated  form  of  sewage.  In  any  case,  it 
is  a  warning  of  possible  pollution,  and  the  determination 
of  free  ammonia,  nitrites,  carbonaceous  matter,  as  shown 
by  "oxygen  consumed,"  and  dissolved  oxygen  yield 
important  evidence  as  to  the  sanitary  quality  of  a  water. 

Furthermore,  nitrates,  the  final  products  of  the  oxida- 
tion of  organic  matter,  and  the  chlorine  introduced  as 
common  salt  into  all  water  which  has  been  in  contact 
with  the  wastes  of  human  life,  furnish  additional  informa- 


172  Elements  of  Water  Bacteriology. 

tion  as  to  the  antecedents  of  a  sample.  The  results  of 
the  chlorine  determination  are  indeed  perhaps  more  clear 
than  those  of  any  other  part  of  the  analysis,  for  chlorine 
and  sewage  pollution  vary  together,  due  allowance  being 
made  for  the  proximity  of  the  sea  and  other  geological 
and  meteorological  factors.  Unfortunately,  it  is  only 
past  history  and  not  present  conditions  which  these  lat- 
ter tests  reveal,  for  in  a  ground-water  completely  puri- 
fied from  a  sanitary  standpoint  such  soluble  constituents 
remain,  of  course,  unchanged.  Thus,  in  the  last  resort, 
it  is  upon  the  presence  and  amount  of  decomposing 
organic  matter  in  the  water  that  the  opinion  of  the 
chemist  must  be  based. 

The  decomposition  of  organic  matter  may  be  measured 
either  by  the  material  decomposed  or  by  the  number  of 
organisms  engaged  in  carrying  out  the  process  of  decom- 
position. The  latter  method  has  the  advantage  of  far 
greater  delicacy,  since  the  bacteria  respond  by  enormous 
multiplication  to  very  slight  increase  in  their  food-supply, 
and  thus  it  comes  about  that  the  standard  gelatin-plate 
count  at  20  degrees  roughly  corresponds,  in  not  too 
heavily  polluted  waters,  to  the  free  ammonia  and  "  oxygen 
consumed,"  as  revealed  by  chemical  analysis.  If  low 
numbers  of  bacteria  are  found,  the  evidence  is  highly 
reassuring,  for  it  is  seldom  that  water  could  be  contami- 
nated under  natural  conditions  without  the  direct  addi- 
tion of  foreign  bacteria  or  of  organic  matter  which 
would  condition  a  rapid  multiplication  of  those  already 


Bacteriological  Examination.  173 

present.  The  bacteriologist  in  such  cases  can  declare 
the  innocence  of  the  water  with  justifiable  certainty. 
When  high  numbers  are  found  the  interpretation  is  less 
simple,  since  they  may  exceptionally  be  due  to  the  multi- 
plication of  certain  peculiar  water  forms.  Large  counts, 
however,  under  ordinary  conditions,  when  including  a 
normal  variety  of  forms  indicate  the  presence  of  an  excess 
of  organic  matter  derived  in  all  probability  either  from 
sewage  or  from  the  fresh  washings  of  the  surface  of  the 
ground.  In  either  case  danger  is  indicated. 

A  still  closer  measure  of  polluting  material  may  be 
obtained  from  the  numbers  of  colonies  which  develop  on 
litmus  lactose  agar  at  37  degrees,  since  organisms  which 
thrive  at  the  body  temperature,  and  particularly  those 
which  ferment  lactose,  are  characteristic  of  the  intestinal 
tract  and  occur  but  rarely  in  normal  waters. 

Gage  (Gage,  1907)  has  shown  that  by  counts  at  20,  30, 
40,  and  50°  C.,  information  may  be  quickly  obtained 
which  is  of  great  assistance  in  judging  the  character  of 
the  water. 

"  Modern  methods  of  bacterial  examination  of  water, 
consisting  usually  of  determinations  of  the  numbers  of 
bacteria  by  means  of  plates  incubated  at  room  tempera- 
ture, and  of  tests  for  the  presence  or  absence  of  one  or  two 
specific  types,  occasionally  lead  to  an  erroneous  interpre- 
tation of  the  quality  of  a  water,  owing  to  the  fact  that  they 
do  not  yield  adequate  data  by  which  abnormal  and  inac- 
curate results  may  be  separated  from  those  which  are 


1/4  Elements  of  Water  Bacteriology. 

truly  indicative  of  purity  or  pollution.  Furthermore,  as 
several  days  must  elapse  before  the  bacterial  tests  can  be 
completed,  the  results  when  obtained  may  have  passed 
their  usefulness.  If,  however,  we  can  so  modify  our 
procedure  that  the  varied  character  of  the  bacteria  in 
waters  of  different  classes  may  be  quickly  and  accurately 
recognized,  the  value  of  bacterial  water  analysis  will  be 
enormously  increased.  Much  of  this  information  may 
be  obtained  by  the  use  of  selective  media,  selective  tem- 
peratures, or  by  a  proper  combination  of  the  two. 

"  By  the  use  of  litmus  lactose  agar  in  place  of  agar  or 
gelatin  we  obtain  similar  counts  of  total  bacteria,  and  in 
addition  are  able  to  separate  those  bacteria  into  two 
groups,  which  do  and  do  not  produce  acid  fermentation 
of  lactose,  and  the  numbers  of  the  two  classes  of  bacteria 
so  obtained  indicate  more  completely  the  character  of  the 
water  than  would  the  numbers  of  either  class  alone. 
By  incubating  our  plates  at  temperatures  of  30  or  40°  C. 
we  are  able  to  obtain  counts  in  twelve  to  eighteen  hours, 
which  counts,  while  smaller  than  those  on  plates  incubated 
for  a  longer  period  at  a  lower  temperature,  appear  to  be 
fully  as  significant.  If  we  increase  our  number  of  deter- 
minations by  incubating  duplicate  plates  at  two  or  more 
temperatures,  the  various  results  and  the  ratios  between 
them  furnish  a  check  upon  one  another  in  addition  to 
increasing  the  available  data  upon  which  to  base  an 
interpretation. 

"  The  distinction  between  polluted  waters  and  waters  of 


Bacteriological  Examination.  175 

good  quality  is  more  sharply  marked  by  counts  at  30°  C. 
than  is  the  case  with  counts  at  20°  C.  Determinations 
at  this  temperature  appear  to  be  especially  applicable  to 
the  control  of  water  niters,  since  the  relative  quality  of 
the  raw  and  filtered  waters  and  the  expression  of  the 
removal  of  bacteria  by  those  filters  are  practically  identical 
with  determinations  made  at  20°  C.,  and,  in  addition, 
they  become  available  within  a  few  hours  after  the  sample 
is  collected."  (Gage,  1907.) 

Finally,  the  search  for  the  Bacillus  coli  furnishes  the 
most  satisfactory  of  all  single  tests  for  fecal  contamination. 
This  organism  is  preeminently  a  denizen  of  the  alimen- 
tary canal  and  may  be  isolated  with  ease  from  waters  to 
which  even  a  small  proportion  of  sewage  has  been  added. 
On  the  other  hand,  it  is  never  found  in  abundance  in 
waters  of  good  sanitary  quality,  and  its  numbers  form 
an  excellent  index  of  the  value  of  waters  of  an  interme- 
diate grade.  The  streptococci  appear  to  be  forms  of  a 
similar  significance  useful  as  yielding  a  certain  amount  of 
confirmatory  evidence.  The  full  bacteriological  analysis 
should  then  consist  of  three  parts  —  the  gelatin-plate  count, 
as  an  estimate  of  the  amount  of  organic  decomposition 
in  process;  the  total  count,  and  the  count  of  red  colonies, 
on  litmus  lactose  agar,  as  a  measure  of  the  organisms 
which  form  acids  and  thrive  at  the  body  temperature; 
and  the  study  of  a  series  of  dextrose-broth  tubes  for  the 
isolation  of  colon  bacilli  and  streptococci.  The  simple 
examination  of  the  lactose-bile  tube  or  the  count  on 


176  Elements  of  Water  Bacteriology. 

the  litmus-lactose-agar  plate  serve  for  what  Whipple  has 
well  called  presumptive  tests. 

The  results  of  the  bacteriological  examination  have,  in 
several  respects,  a  peculiar  and  unique  significance. 
First,  this  examination  is  the  most  direct  method  of  sani- 
tary water  analysis.  The  occurrence  of  nitrites  or  free 
ammonia  in  a  small  fraction  of  one  part  per  million,  or  of 
chlorine  in  several  parts  per  million,  do  not  in  themselves 
render  a  water  objectionable  or  dangerous.  They  merely 
serve  as  indicators  to  show  that  germ-containing  and 
germ-sustaining  organic  matter  is  present.  By  a  determi- 
nation of  the  chlorine  and  study  of  the  relations  of  carbon 
and  nitrogen,  it  is  possible  to  determine  with  some  degree 
of  accuracy  whether  this  organic  matter  is  of  plant  or 
animal  origin,  and  hence  to  rate  its  objectionable  or  dan- 
gerous character.  By  the  bacteriological  examination,  on 
the  other  hand,  we  are  able  to  determine  directly  whether 
particular  kinds  of  organisms  characteristic  of  sewage  are, 
or  are  not,  actually  present  in  the  water.  What  we  dread 
in  drinking-water  is  the  presence  of  pathogenic  bacteria, 
mainly  from  the  intestinal  tract  of  man,  and  it  is  quite 
certain  that  the  related  non-pathogenic  bacteria  from  the 
same  source  will  behave  more  nearly  as  these  disease 
germs  do  than  will  any  chemical  compounds.  In  the 
second  place,  the  bacteriological  methods  are  superior 
in  delicacy  to  any  others.  Klein  and  Houston  (1898) 
showed  by  experiment  with  dilutions  of  sewage  that 
the  colon  test  was  from  ten  to  one  hundred  times  as 


Bacteriological  Examination.  177 

sensitive  as  the  methods  of  chemical  analysis;  and  studies 
of  the  self-purification  of  streams  have  confirmed  their 
results  on  a  practical  scale.  Thus  in  the  Sudbury  River 
it  was  found  that  while  the  chemical  evidences  of 
pollution  persisted  for  six  miles  beyond  the  point  of 
entrance,  the  bacteria  introduced  could  be  detected  for 
four  miles  further  (Woodman,  Winslow,  and  Hansen, 
1902). 

The  statement  is  sometimes  made  that  while  bac- 
teriological methods  may  be  more  delicate  for  the  detec- 
tion of  pollution  in  surface-waters,  contamination  in 
ground-waters  may  best  be  discovered  by  the  chemical 
analysis.  That  such  is  not  the  case  has  been  well  shown 
by  Whipple  (Whipple,  1903),  who  cites  the  following  two 
instances  in  which  the  presumptive  test  revealed  con- 
tamination not  shown  by  the  chemical  analysis: 

"  A  certain  driven- well  station  was  located  in  swampy 
land  along  the  shores  of  a  stream,  and  the  tops  of  the 
wells  were  so  placed  that  they  were  occasionally  flooded 
at  times  of  high  water.  The  water  in  the  stream  was 
objectionable  from  the  sanitary  standpoint.  The  wells 
themselves  were  more  than  100  feet  deep;  they  pene- 
trated a  clay  bed  and  yielded  what  may  be  termed  arte- 
sian water.  Tests  for  the  presence  of  Bacillus  coli  had 
invariably  given  negative  results,  as  might  be  naturally 
expected.  Suddenly,  however,  the  tests  became  positive 
and  so  continued  for  several  days.  On  investigation  it 
was  found  that  some  of  the  wells  had  been  taken  up  to 


178  Elements  of  Water  Bacteriology. 

be  cleaned,  and  that  the  workmen  in  resinking  them  had 
used  the  water  of  the  brook  for  washing  them  down. 
This  allowed  some  of  the  brook-water  to  enter  the  system. 
It  was  also  found  that  at  the  same  time  the  water  in  the 
brook  had  been  high,  and  because  of  the  lack  of  packing 
in  certain  joints  at  the  top  of  the  wells,  the  brook-water' 
leaked  into  the  suction  main.     The  remedy  was  obvious 
and  was  immediately  applied,  after  which  the  tests  for 
Bacillus   coli  once   more  became  negative.     During   all 
this  time  the  chemical  analysis  of  the  water  was  not  suffi- 
ciently abnormal  to  attract  attention.     On  another  occa- 
sion a  water-supply  taken  from  a  small  pond  fed  by 
springs,   and  which  was  practically  a  large  open  well, 
began  to  give  positive  tests  for  Bacillus  coli,  and  on  exami- 
nation it  was  found  that  a  gate  which  kept  out  the  water 
of  a  brook  which  had  been  formerly  connected  with  the 
pond  was  open  at  the  bottom,  although  it  was  supposed 
to  have  been  shut,  thus  admitting  a  contaminated  sur- 
face-water to  the  supply."     Whipple  also  calls  attention 
to  the  report  on  the  Chemical  and  Bacteriological  Exami- 
nation of  Chichester  Well-waters  by  Houston  (Houston, 
1901),  in  which  the  results  of  chemical  and  bacteriologi- 
cal examinations  of  thirty  wells  were   compared.     It  was 
found   that   the   bacteriological   results   were   in   general 
concordant  and  satisfactory.     The  wells  which  were  high- 
est in  the  number  of  bacteria  showed  also  the  greatest 
amount  of  pollution,  as  indicated  by  the  numbers  of  B. 
coli,  B.  sporogenes,  and  streptococci.     On  the  other  hand, 


Bacteriological  Examination. 


the  chlorine  and  the  albuminoid  ammonia  showed  no 
correspondence  with  the  bacteriological  results. 

Vincent  (Vincent,  1905)  cites  an  interesting  case  of 
the  detection  of  progressive  pollution  of  a  ground-water 
by  bacteriological  methods.  The  well  of  a  military  camp 
in  Algeria  showed  200  bacteria  per  c.c.  before  the  arrival 
of  a  regiment  of  troops.  Its  subsequent  history  is  indi- 
cated in  the  table  below. 

PROGRESSIVE  POLLUTION   OF  A  WELL. 

(VINCENT,  1005.) 


» 

Bacteria 
per  c.c. 

Bacillus  Coli 
per  c.c. 

Before  arrival  of  troops 

200 

O 

6  days  after  arrival   .    . 

77O 

O 

14  days  after  arrival      

4240 

I 

41  days  after  arrival     

6060 

2 

60  days  after  arrival 

Id  OOO 

IO 

Thirdly,  negative  tests  for  Bacillus  coli  and  low  bac- 
terial counts  may  be  interpreted  as  proofs  of  the  good 
quality  of  water,  with  a  certainty  not  attainable  by  any 
other  method  of  analysis.  Many  a  surface-water  with 
reasonably  low  chlorine  and  ammonias  has  caused  epi- 
demics of  typhoid  fever;  but  it  is  impossible,  under  any 
natural  conditions,  that  a  water  could  contain  the  typhoid 
bacillus  without  giving  clear  evidence  of  pollution  in  the 
dextrose-broth  tube  or  on  the  lactose-agar  plate. 

In  the  examination  of  springs,  especially  those  used  for 
domestic  supplies  at  country  houses,  the  authors  have 


180  Elements  of  Water  Bacteriology. 

found  that  the  bacteriological  examination  offers  a  more 
delicate  and  a  more  certain  index  of  the  quality  than 
may  be  obtained  by  chemical  analysis.  In  a  number  of 
instances,  springs  located  in  pastures  have  become  slightly 
polluted  by  animals,  but  to  so  small  an  extent  that  the 
chemical  examination  gave  no  indication  of  trouble. 
The  bacteria,  however,  increased  greatly  in  number,  and 
colon  bacilli  could  be  readily  isolated  from  75  per 
cent  of  the  i-c.c.  samples  of  a  long  series  used  in 
making  the  presumptive  test.  A  single  case  may  suffice 
as  an  illustration.  This  was  a  spring  located  on  a  hill  in 
Hopkinton,  Mass. 
The  chemical  analysis  was  as  follows: 

Color None 

Turbidity None 

Sediment None 

Odor  (hot) None 

Odor  (cold) None 

Parts  per  Million. 

Total  solids 33-oooo 

Loss  on  ignition 7.0000 

Fixed  residue 26.0000 

Hardness f 11.0000 

Chlorine * 10.0000 

Nitrogen  as  — 

albuminoid  ammonia      o.oooo 

free o.oooo 

nitrites o.oooo 

nitrates o.oooo 

The  bacteriological  examination  showed  a  total  count 
of  375  bacteria  per  c.c.  and  a  37-degree  count  of  350 
per  c.c. 


Bacteriological  Examination.  181 

The  presumptive  tests  for  Bacillus  coli  showed  that  gas- 
producing  organisms  were  present  in  a  majority  of  i-c.c. 
samples,  and  typical  colon  bacilli  were  isolated.  In  this 
case  the  contamination  was  brought  about  by  cattle  gam- 
ing access  to  the  area  immediately  surrounding  the  spring; 
but  the  same  conditions  might  easily  have  led  to  infec- 
tion from  human  beings. 

Similar  results  have  been  reported  by  Savage  and 
Bulstrode  (Savage,  1906)  in  the  examination  of  the  water- 
supply  of  Bridgend. 

It  seems  to  the  writers  that  the  real  application  of 
chemistry  begins  where  that  of  bacteriology  ends.  When 
pollution  is  so  gross  that  its  existence  is  obvious  and  only 
its  amount  needs  to  be  determined,  the  bacteriological 
tests  will  not  serve,  on  account  of  their  excessive  delicacy. 
In  studying  the  heavy  pollution  of  small  streams,  the 
treatment  of  trades  wastes,  and  the  purification  of  sewage, 
the  relations  of  nitrogenous  compounds  and  of  oxygen 
compounds  are  of  prime  importance.  In  other  words, 
when  pollution  is  to  be  avoided,  because  the  decompo- 
sition of  chemical  substances  causes  a  nuisance,  it  must 
be  studied  by  chemical  methods.  When  the  danger  is 
sanitary  and  comes  only  from  the  presence  of  bacteria, 
bacteriological  methods  furnish  the  best  index  of  pollution. 

In  the  study  of  certain  special  problems  the  paramount 
importance  of  bacteriology  is  generally  recognized.  The 
distribution  of  sewage  in  large  bodies  of  water  into  which 
it  has  been  discharged  may  thus  best  be  traced  on  account 


1 82  Elements  of  Water  Bacteriology. 

of  the  ready  response  of  the  bacterial  counts  to  slight 
proportions  of  sewage,  particularly  since  the  ease  and 
rapidity  with  which  the  technique  of  plating  can  be  carried 
out  make  it  possible  to  examine  a  large  series  of  samples 
with  a  minimum  of  time  and  trouble.  The  course  of  the 
sewage  carried  out  by  the  tide  from  the  outlet  of  the 
South  Metropolitan  District  of  Boston  was  studied  in 
this  way  by  E.  P.  Osgood  in  1897,  and  mapped  out  by 
its  high  bacterial  content  with  greater  accuracy  than 
could  be  attained  by  any  other  method.  Some  very 
remarkable  facts  have  been  developed  by  similar  studies 
as  to  the  persistence  of  separate  streams  of  water  in 
immediate  contact  with  each  other.  Heider  showed  that 
the  sewage  of  Vienna,  after  its  discharge  into  the  Danube 
River,  flowed  along  the  right  bank  of  the  stream,  pre- 
serving its  own  bacterial  characteristics  and  not  mixing 
perfectly  with  the  water  of  the  river  for  a  distance  of 
more  than  twenty-four  miles  (Heider,  1893).  Jordan 
(Jordan,  1900),  in  studying  the  self-purification  of  the 
sewage  discharged  from  the  great  Chicago  drainage 
canal,  found  by  bacteriological  analyses  that  the  Des 
Plaines  and  the  Kankakee  Rivers  could  both  be  distin- 
guished flowing  along  in  the  bed  of  the  Illinois,  the  two 
streams  being  in  contact,  yet  each  maintaining  its  own 
individuality.  Finally,  the  quickness  with  which  slight 
changes  in  the  character  of  a  water  are  marked  by  fluc- 
tuations in  bacterial  numbers  renders  the  bacteriological 
methods  invaluable  for  the  daily  supervision  of  surface 


Bacteriological  Examination.  183 

supplies   or  of   the   effluents   from   municipal   nitration 
plants. 

In  the  commoner  case,  when  normal  values  obtained 
by  such  routine  analyses  are  not  at  hand,  the  problem  of 
the  interpretation  of  any  sanitary  analysis  is  a  more  diffi- 
cult one.  The  conditions  which  surround  a  source  of 
water-supply  may  be  constantly  changing.  No  engineer 
can  measure  the  flow  of  a  stream  in  July  and  deduce  the 
amount  of  water  which  will  pass  in  February;  yet  the 
July  gauging  has  its  own  value  and  significance.  So  a 
single  analysis  of  any  sort  is  not  sufficient  for  all  past  and 
future  time.  If  it  gives  a  correct  picture  of  the  hygienic 
condition  of  the  water  at  the  moment  of  examination  it 
has  fulfilled  its  task,  and  this  the  bacteriological  analysis 
can  do.  The  evidence  furnished  by  inspection  and  by 
chemical  analysis  should  be  sought  for  and  welcomed 
whenever  it  can  be  obtained,  yet  we  are  of  the  opinion 
that,  on  account  of  their  directness,  their  delicacy,  and 
their  certainty,  the  bacteriological  methods  should  least 
of  all  be  omitted,  and,  if  necessary,  they  alone  may  fur- 
nish conclusive  testimony  as  to  the  safety  of  a  potable 
water. 


CHAPTER    XI. 

BACTERIOLOGY  OF  SEWAGE  AND   SEWAGE  EFFLUENTS. 

THE  first  object  of  modern  sewage  disposal  is  the 
oxidation  of  putrescible  organic  matter.  Chemical,  rather 
than  bacterial,  purification  is  the  prime  requisite;  and 
chemical  tests  therefore  serve  best  as  criteria  of  the  results 
obtained.  Bacteria  are  the  agents  in  the  process  of  sewage 
purification;  but  the  most  generally  useful  measure  of  the 
work  accomplished  is  the  chemical  oxidation  attained. 
"To  employ  a  simile,  it  is  a  case  of  the  saw  and  the 
two-foot  rule  —  the  saw  will  do  the  cutting,  but  the 
rule  will  measure  the  work  cut."  (W.  J.  Dibdin.) 

In  certain  cases,  however,  bacterial  as  well  as  chemical 
purity  must  be  effected,  in  view  of  special  local  require- 
ments. The  sewage  from  a  contagious  disease  hospital, 
for  example,  should  be  freed  from  infectious  material  as 
a  factor  of  safety.  Sewage  discharged  into  a  body  of 
water  adapted  for  bathing  may  well  be  so  treated  as  to 
protect  those  using  the  water.  In  the  case  of  seaboard 
cities  where  sewage  effluents  are  likely  to  contaminate 
oyster  beds  and  other  layings  of  edible  shellfish  the  prob- 
lem assumes  great  importance.  Where  bacterially  im- 

184 


Sewage  and  Sewage  Effl^^ents.  18$ 

pure  effluents  are  discharged  into  streams  used  for  sources 
of  water-supply  the  town  taking  water  may  protect  itself 
by  filtration.  It  should  so  protect  itself,  at  any  rate, 
from  the  pollution  necessarily  incident  to  surface-waters; 
and,  unless  the  bacterial  condition  of  a  stream  or  lake  is 
made  very  materially  worse  by  the  discharge  of  sewage 
effluents,  it  is  fair  that  the  responsibility  of  purification 
should  rest  on  the  water  works,  rather  than  on  the  sewage 
purification  plant.  Shellfish,  on  the  other  hand,  cannot 
be  purified.  Either  pollution  must  be  prevented,  or  the 
industry  abandoned.  Under  such  circumstances  sanitary 
authorities  may  rightly  demand,  as  they  have  demanded 
at  Baltimore,  that  bacteria,  as  well  as  putrescible  organic 
matter,  shall  be  removed  in  sewage  treatment.  Under 
such  circumstances  the  bacterial  control  of  purification 
plants  is  as  essential  as  in  the  case  of  water  filters. 

In  England,  considerable  attention  has  been  devoted  to 
this  subject  and  numerous  methods  have  been  recom- 
mended as  furnishing  valuable  criteria  of  the  bacterial 
quality  of  sewage  effluents.  Houston  (i902b),  for 
example,  suggests  various  tests  involving  the  use  of  litmus 
milk,  pepton  solution,  gelatin  tubes,  and  neutral-red 
broth,  as  well  as  the  inoculation  of  animals.  He  con- 
siders the  determination  of  the  numbers  of  B.  coli  and 
B.  sporogenes  as  of  greatest  moment,  while  the  identifi- 
cation of  streptococci  is  of  value  in  certain  cases,  and  the 
enumeration  of  liquefying  bacteria,  spore-forming  aerobes, 
thermophilic  bacteria,  and  hydrogen  sulphide  producing 


1 86  Elements  of  Water  Bacteriology. 

bacteria  is  of  subsidiary  importance.  Rideal  (1906)  has 
recently  commended  a  somewhat  less  extensive  series  o'f 
tests,  including  aerobic  and  anaerobic  counts,  both  at  20 
and  37  degrees,  with  the  determination  of  the  number  of 
liquefiers  and  the  number  of  spore  formers.  The  results 
attained  do  not  seem  to  warrant  any  such  elaborate  pro- 
cedure. As  far  as  the  authors  are  aware,  the  determination 
of  liquefying  bacteria,  anaerobic  bacteria  and  thermo- 
philic  bacteria  does  not  add  any  information  of  material 
importance  to  that  obtained  from  the  total  count.  Some 
test  for  specific  sewage  organisms  is  of  course  desirable. 
Here  again,  however,  the  determination  of  B.  sporogenes 
and  sewage  streptococci  tells  the  observer  little  more  than 
can  be  learned  from  the  routine  use  of  the  colon  test.  In 
the  United  States  the  practice  of  sewage  bacteriologists  is 
crystallizing  around  the  total  count  and  the  estimation  of 
B.  coli.  In  the  absence  of  evidence  as  to  the  specific 
value  of  other  data,  the  routine  control  of  filter  plants 
may  well  be  limited  to  these  two  determinations. 

The  total  count  of  bacteria  should  be  made,  as  in  the 
case  of  waters,  on  gelatin  at  20  degrees.  Determinations 
carried  out  in  duplicate  on  agar  at  37  degrees  add  addi- 
tional information  of  considerable  value.  The  ratio  of 
the  37-degree  count  to  the  20-degree  count  varies  with 
different  sewages.  At  Boston  the  body  temperature  count 
is  70  to  80  per  cent  of  the  total  count;  at  Lawrence  it 
appears  to  be  proportionately  much  lower  (Gage,  1906). 
In  using  either  medium,  it  is  well  to  add  lactose  and  lit- 


Sewage  and  Sewage  Effluents.  187 

mus  and  note  the  number  of  red  colonies,  as  a  check  on 
the  enumeration  of  B.  coli. 

The  determination  of  the  number  of  colon  bacilli  in 
sewage  and  effluents  should  furnish  an  integral  part  of 
bacteriological  sewage  analysis  since  it  is  important  to 
know  whether  the  decrease  of  intestinal  bacteria  in  the 
process  of  purification  is  proportional  to  the  reduction  of 
total  bacteria.  The  State  Sewerage  Commission  of  New 
Jersey  has  adopted  this  procedure  in  its  supervision  of 
the  disposal  plants  in  that  state;  and  the  results  seem 
amply  commensurate  with  the  labor  involved.  As  in  the 
case  of  polluted  waters  the  enumeration  of  B.  coli  may  be 
carried  out,  either  by  the  study  of  the  red  colonies  which 
appear  on  litmus-lactose-agar  plates  inoculated  with  the 
sample  directly,  or  by  the  use  of  a  preliminary  enrich- 
ment process.  The  complete  identification  of  B.  coli 
seems  unnecessarily  tedious,  however,  where  the  organ- 
isms are  present  in  such  abundance.  Some  approximate 
presumptive  method  is  indicated  here,  if  anywhere;  and  the 
experience  with  polluted  water,  reviewed  in  Chapter  VIII, 
points  to  the  Jackson  bile  medium  as  the  most  prom- 
ising one.  More  than  a  year's  experience  at  the  Sewage 
Experiment  Station  of  the  Massachusetts  Institute  of 
Technology  has  shown  that  this  presumptive  test  in  general 
yields  good  results.  As  pointed  out  above,  a  72-hour 
incubation  period  at  37  degrees  is  required.  All  tubes 
showing  20  per  cent  gas  at  the  end  of  this  time  may  be 
considered  positive  tests  for  B.  coli  without  serious  error. 


1 88  Elements  of  Water  Bacteriology. 

The  total  number  of  bacteria  and  the  number  of  colon 
bacilli  naturally  vary  widely  in  the  sewages  of  different 
cities  and  towns.  European  sewages,  being  more  con- 
centrated, show  as  a  rule  higher  numbers  than  are  found 
in  America.  Results  compiled  from  various  sources 
show  from  one  to  five  million  bacteria  in  the  sewages  of 
Essen,  Berlin,  Charlottenburg,  Leeds,  Exeter,  Chorley, 
and  Oxford,  two  to  ten  millions  in  the  sewages  of  Lon- 
don, Walton  and  W.  Derby,  and  over  ten  millions  in  the 
sewages  of  Paris,  Ballater  and  Belfast  (Winslow,  1905). 
The  number  of  colon  bacilli  in  English  sewages  varies 
from  50,000  to  750,000.  In  American  sewages,  on  the 
other  hand,  bacteria  are  somewhat  less  numerous.  At 
Lawrence  the  determinations  made  from  1894  to  1901 
showed  on  the  average  2,800,000  bacteria  per  c.c.  At 
Worcester,  Eddy  reported  3,712,000  in  1901  (Eddy, 
1902);  at  Ames,  Iowa,  Walker  (1901)  found  1,248,256 
in  the  same  year.  At  Columbus,  Johnson  (1905)  reports 
an  average  of  3,600,000  bacteria  per  c.c.;  the  individual 
numbers  varied  from  320,000  to  27,000,000.  The  num- 
ber of  colon  bacilli  varied  from  50,000  to  1,000,000  and 
averaged  500,000.  Day  samples  of  Boston  sewage  col- 
lected three  times  a  week,  from  October,  1906,  to  April, 
1907,  showed  an  average  of  1,200,000  bacteria  per  c.c. 
In  the  summer  months  numbers  are  notably  higher  than 
at  other  seasons  in  many  sewages.  Thus  in  1903,  Boston 
sewage  contained  2,995,000  bacteria  in  July,  4,263,600  in 
August,  11,487,500  in  September,  3,693,000  in  October, 


Sewage  and  Sewage  Effluents.  189 

587,100  in  November,  and  712,000  in  December  (Wins- 
low,  1905).  There  is  also  a  marked  diurnal  variation 
in  the  bacterial  content  of  sewage,  since  the  flow  contains 
a  smaller  proportion  of  intestinal  matter  at  night  than 
at  other  times.  For  example,  a  series  of  hourly  samples 
at  the  Sewage  Experiment  Station  of  the  Massachusetts 
Institute  of  Technology  showed  the  following  results : 

BACTERIA  IN  BOSTON  SEWAGE  —  AVERAGES    FOR    EACH 

4-HOUR  PERIOD.     AUGUST   13-14,  1903. 

(WINSLOW  AND  PHELPS,  1905.) 


Period. 

Bacteria  per  c.c. 

7  3O—  II  3O  A  M 

i  800  ooo 

11.30  A.M.-3-30  P.M  

•2  ?o—  7  7O  P  M. 

3,200,000 
4,600,000 

7  20—  II.  3O  P.M. 

•?,";  00,000 

11.30  P.M.—  3.30  A.M  

1,000,000 

3.3O—  7.3O  A.M. 

400,000 

It  is  evident  that  many  published  results  of  bacterial 
examinations  of  sewage  are  in  excess  of  the  true  values, 
since  they  refer  in  most  cases  to  day  samples  only. 

The  bacterial  content  of  sewage  effluents  varies  widely 
according  to  the  process  of  purification  adopted  and  the 
efficiency  of  the  particular  plant.  The  only  process 
which  yields  a  notably  purified  effluent  from  the  bacteri- 
ological standpoint  is  that  of  filtration  through  sand. 
Processes  of  this  type  when  operated  with  care  may  give 
a  bacterial  purification  well  over  99  per  cent.  The  aver- 
age numbers  obtained  from  the  outdoor  experimental 
niters  at  Lawrence  (each  •&-$  acre)  in  1905  are  tabu- 
lated below. 


I90 


Elements  of  Water  Bacteriology. 


BACTERIA   IN    SEWAGE   AND    SAND    FILTER   EFFLUENTS 

AT  LAWRENCE. 

(CLARK,  1906.) 

Bacteria  per  c.c. 


Applied  sewage  .  . 
Filter  i 

1,206,000 
8  ?it; 

Filter  $C  .... 
Filter  6 

2,906 
10  896 

Filter  2  
Filter  4 

1,059 

r87 

Filter  9A  .... 
Filter  10  

4,585 

I    747 

Filter  56  .... 

19,200 

The  Septic  Tank  and  Intermittent  Sand  Filter  at  Iowa 
State  College  gave  the  following  bacterial  results  in  1899 
and  1900,  the  averages  representing  daily  examinations 
of  the  sand  effluent  and  weekly  examinations  of  crude 
and  septic  sewage. 

BACTERIA    IN    SEWAGE    SEPTIC    EFFLUENT    AND    SAND 

FILTER  EFFLUENT  AT  IOWA   STATE   COLLEGE. 

(WALKER,  1901.) 


Bacteria  per  c.c. 


Monthly  Averages. 


Month. 

Sewage. 

Septic  Effluent. 

Sand  Effluent. 

August,  1899  .  . 
September  .  .  . 
October  .... 
November  .  .  . 
December  .  .  . 
January,  1900  . 
February  .  .  . 
March  .... 
April  

2,392,600 
8,815,000 
6,064,800 

4,537.333 
816,333 
848,000 
345,533 
I32,i25 
2,121,000 

1,388,300 
3,245,000 
4,941,000 
3,014,000 
848,000 
726,000 
233,8rb 
112,500 
1,392,800 

2,246 
3,66o 

4,320 
2,261 

2,3^9 
830 

3>45l 
2,480 
13,263 

May 

I  O2  I,OOO 

783,300 

3,077 

1,318,100 

1,391,300 

2,359 

Tulv 

3,008,700 

4,578,333 

2,270 

J  "v       •    • 
August     .... 
September  .    .    . 

403,118 
1,181,533 

215,700 
383,733 

546 
850 

Sewage  and  Sewage  Effluents. 


191 


Such  high  efficiencies  as  these  two  tables  indicate  can 
scarcely  be  expected  even  with  the  sand  process  under 
the  actual  working  conditions  of  a  municipal  plant.  At 
Vineland,  N.  J.,  for  example,  the  intermittent  niters  show 
a  reduction  of  90  to  95  per  cent  in  total  bacteria  and  a 
somewhat  higher  reduction  of  B.  coli.  The  results  of 
three  examinations  made  in  1906  are  given  below. 


BACTERIA    IN    SEWAGE    AND    SAND    FILTER    EFFLUENT 
AT   VINELAND,   N.  J. 

(N.  J.  STATE  SEWERAGE  COMMISSION,  1907.) 


Bacteria  per  c.c. 

B.  Coli  in 

Date. 

Sewage. 

Effluent. 

Sewage. 

Effluent. 

March  2  .  . 
July  26  ... 
July  26  ... 

480,000 
496,000 
511,000 

20,000 
61,000 
38,000 

.0001   C.C. 

.0001  c.c. 
.00001  c.c. 

.01  C.C. 

.001  c.c. 
.001  c.c. 

At  Columbus  the  experimental  sand  niters  effected  an 
average  reduction  of  87  per  cent  in  total  bacteria  and  of 
98.5  per  cent  in  colon  bacilli.  The  number  of  B.  coli 
remaining  in  the  effluent  varied  from  500  to  10,000  per 
c.c.  (Johnson,  1905). 


Elements  of  Water  Bacteriology. 


McGowan,  Houston,  and  Kershaw  (1904)  report  the 
following  figures  for  English  sewage  farms. 

ANALYSIS   OF  EFFLUENTS    FROM   SEWAGE   FARMS. 
(McGOWAN,   HOUSTON,   AND   KERSHAW,    1904.) 


Bacteria  per  c.c. 

Place. 

Gelatin,  20°. 

Agar,  37°. 

B.  Coli 

Number. 

Per  Cent 

Removal. 

Number. 

Per  Cent 
Removal. 

Aldershot     . 

183,266 

99 

37,3°8 

99 

1000—10,000 

Altrincham 

363,4°° 

97 

7,275 

99 

IOO-IOOO 

Cambridge  . 

711,476 

94 

78,327 

94 

1000—10,000 

Croydon  .    . 

1,413,200 

95 

112,000 

97 

1000—10,000 

Leicester 

532,777 

95 

70,500 

95 

1000—10,000 

S.  Norwood 

778,322 

98 

35,157 

99 

100-1,000 

Rugby    ;/  , 

637»!33 

97 

81,526 

97 

1000-10,000 

The  newer  bacterial  processes,  contact  beds,  and  trick- 
ling filters  naturally  show  a  much  less  satisfactory 
bacterial  removal  than  sand  filtration  beds.  In  the 
Columbus  experiments,  Johnson  (1905)  found  from  one 
to  two  million  bacteria  in  the  effluents  of  contact  beds 
and  from  750,000  to  1,900,000  in  the  effluents  from 
trickling  filters.  The  average  percentage  reduction  effected 
by  seven  contact  beds  and  six  trickling  filters  is  shown 
below. 


Sewage  and  Sewage  Effluents. 


193 


REDUCTION    OF   BACTERIA   AT   COLUMBUS,    OHIO. 

(JOHNSON,    1905.) 


Contact  Beds. 

Per  Cent 
Reduction. 

Trickling 
Filters. 

Per  Cent 
Reduction. 

Primary  A     

60 

A 

74 

Primary  B     

47 

B 

7O 

Primary  C     

T.T. 

c 

70 

Primary  D 

•?•? 

D 

60 

Primary  E                 .... 

o 

E 

46 

Secondary  A         

*8 

F 

21 

Secondary  B 

IQ 

Thumm  and  Pritzkow  (1903),  at  the  Berlin  Experi- 
ment Station    obtained  the  results  tabulated  below. 


BACTERIA  IN  SEWAGE,  CONTACT  EFFLUENT,  AND  SAND 
EFFLUENT  AT   BERLIN. 


Bacteria 
per  c.c. 

Crude  sewage     

16,900,000 

Primary  contact  effluent  (coarse  coke) 

12,400,000 

Secondary  contact  effluent  (fine  coke) 

5,600,000 

Tertiary  sand  effluent                            

1,100,000 

Primary  contact  effluent  (fine  coke)  
Secondary  sand  effluent 

7,400,000 
1,800,000 

At  the  experiment  station  of  La  Madeleine,  in  Lille, 
Calmette  (1907)  reports  5,000,000  bacteria  per  c.c.  in 
the  crude  sewage,  2,900,000  in  the  second  contact  effluent 
and  800,000  in  the  effluent  from  the  trickling  bed.  Of 
20,000  B.  coli  per  c.c.  applied  to  the  niters,  the  contact 
system  delivered  4000  and  the  trickling  bed  3000  per  c.c. 
The  average  results  of  examinations  made  three  times 


194 


Elements  of  Water  Bacteriology. 


a  week  at  the  Sewage  Experiment  Station  of  the  Massa- 
chusetts Institute  of  Technology,  during  two  different 
periods,  were  as  follows: 

BACTERIA  IN  SEWAGE,  SEPTIC  EFFLUENT,  AND  TRICK- 
LING  EFFLUENTS  AT  BOSTON. 

(WINSLOW   AND  PHELPS,   1907.) 


B.  Coli. 

Bacteria  per  c.c. 

Positive  Tests 

in  .000001  c.c.1 

July  -Sept.,  1906. 

Oct.,  1  9  06  -April, 
1907. 

July-Sept., 
1906. 

Per  Cent 

Per  Cent 

No. 

Reduc- 

No. 

Reduc- 

Per Cent. 

tion. 

tion. 

Sewage  .    .    . 
Septic   effluent 

1,300,000 
1,650,000 

Inc. 

1,200,000 
750,000 

38 

65 
66 

Effluent   from 

trickling      bed 
Septic  tank  and 

750,000 

42 

200,000 

83 

35 

trickling  bed 

750,000 

42 

l8o,000 

85 

35 

1  Jackson  bile  test. 

The  contact  beds,  as  operated  on  a  practical  scale  in 
England,  show  considerably  higher  numbers.  At  Lon- 
don the  Barking  and  Crossness  beds  yielded  effluents 
containing  one  to  five  million  bacteria  per  c.c.,  of  which 
100,000  to  600,000  were  B.  coli. 

There  are  few  plants  of  the  newer  types  now  in  opera- 
tion in  the  United  States,  and  fewer  still  are  controlled 
by  bacteriological  examinations.  At  Plainfield,  N.  J., 


Sewage  and  Sewage  Effluents. 


195 


however,  the  combination  of  septic  tank  and  double 
contact  beds  produces  a  bacterial  purification  of  80  to 
90  per  cent  as  measured  by  total  numbers.  The  fol- 
lowing table  shows  the  results  of  four  examinations 
made  in  1906. 

BACTERIA    IN    SEWAGE,    SEPTIC    EFFLUENT,   AND    CON- 
TACT  EFFLUENT  AT  PLAINFIELD,   N.  J. 
(N.J.  STATE  SEWERAGE  COMMISSION,   1907.) 


Date. 

Bacteria  per  c.c. 

B.  Coli  in  — 

Sewage. 

Septic 
Effluent. 

Secondary 
Contact 
Effluent. 

Sewage. 

Septic 
Effluent. 

Secondary 
Contact 
Effluent. 

c.c. 

c.c. 

c.c. 

July  9  • 
July  9  • 

Aug.  9. 
Aug.  9  . 

2,295,200 
2,043,300 
1,371,700 
1,655,000 

659,200 

555>oo° 
989,700 

SQi^oo 
172,600 
186,700 
338,000 

.00001 
.  OOOOOI 
.00001 
.  OOOOOI 

.0000  i 
.0001 
.0001 

.0001 
.0001 

.0001 
.00001 

It  is  obvious  that  effluents  of  this  character  cannot  be 
considered  satisfactory  from  the  standpoint  of  bacterial 
purification.  As  Houston  concluded,  after  a  careful 
review  of  the  subject,  "The  different  kinds  of  bacteria 
and  their  relative  abundance  appear  to  be  very  much  the 
same  in  the  effluents  as  in  the  crude  sewage.  Thus,  as 
regards  undesirable  bacteria,  the  effluents  frequently  con- 
tain nearly  as  many  B.  coli,  proteus-like  germs,  spores  of 
B.  enteritidis  sporogenes  and  streptococci,  as  crude  sew- 
age. In  no  case,  seemingly,  has  the  reduction  of  these 
objectionable  bacteria  been  so  marked  as  to  be  very 


196  Elements  of  Water  Bacteriology. 

material  from  the  point  of  view  of  the  epidemiologist  " 
(Houston,  i902a). 

Experimental  studies  with  specific  bacteria  have  con- 
firmed these  conclusions.  Houston  (1904^  found  that 
B.  pyocyaneus  appeared  in  the  effluent  of  a  trickling  bed 
ten  minutes  after  application  to  the  top  and  continued  to 
be  discharged  for  ten  days.  In  septic  tanks  and  contact 
beds,  the  same  germ  persisted  for  ten  days.  Rideal 
(1906)  quotes  experiments  by  Pickard  at  Exeter,  which 
show  that  typhoid  bacilli  may  persist  for  two  weeks  in  a 
septic  tank  and  that  contact  bed  treatment  only  effects  a 
90  per  cent  removal  of  these  organisms. 

Where  bacterial  purity  is  required  some  special  process 
of  disinfection  must  be  combined  with  the  contact  bed 
or  the  trickling  filter.  For  this  purpose  treatment  with 
chloride  of  lime  or  other  chemicals  is  rapidly  gaining 
ground  as  an  important  adjunct  to  bacterial  disposal 
plants;  and  in  connection  with  this  process  bacteriological 
control  is  an  essential. 

Rideal  (1906)  first  showed  at  Guildford  that  30  parts 
of  available  chlorine  per  million  would  reduce  the  number 
of  bacteria  in  crude  sewage  from  several  millions  to 
50,000,  while  50  parts  would  reduce  their  number  to  20 
per  c.c.  Colon  bacilli  were  reduced  from  one  million 
per  c.c.  to  less  than  one  per  c.c.  by  30  parts  of  chlorine. 
In  septic  effluent  25  to  44  parts  of  chlorine  per  million 
reduced  B.  coli  from  two  and  a  half  to  four  and  a  half 
million  per  c.c.  to  less  than  one  per  c.c.  With  contact 


Sewage  and  Sewage  Effluents. 


197 


effluents  smaller  amounts  of  chlorine  proved  efficient. 
The  primary  effluent  required  20  parts  per  million,  the 
secondary  effluent  10.6  parts  per  million  and  the  tertiary 
effluent  2.5  parts  per  million  to  reduce  the  number  of 
B.  coli  so  that  this  organism  could  not  be  isolated  in  5  c.c. 
In  this  country  Phelps  and  Carpenter  (1906)  demon- 
strated the  practical  usefulness  of  bleaching  powder 
disinfection,  at  the  Sewage  Experiment  Station  of  the 
Massachusetts  Institute  of  Technology.  As  indicated  in 
the  table  below,  smaller  amounts  of  chlorine  than  were 
used  by  Rideal  will  give  good  results  with  more  dilute 
American  sewages. 

BACTERIA    IN    TRICKLING    FILTER    EFFLUENT    BEFORE 
AND    AFTER  TREATMENT   WITH    CHLORIDE  OF  LIME 
(5    PARTS    PER    MILLION    AVAILABLE    CHLORINE). 
(PHELPS  AND  CARPENTER,  1906.) 


Bacteria  per  c.c. 

B.  Coli,  Jackson  Bile  Test. 

Date. 

Before. 

After. 

Before 

After  i.o  c.c. 

.000001  c.c. 

1906. 

August  ii.. 

270,000 

69 

+       0 

+       0 

13-  • 

630,000 

41 

0        0 

+     o 

14.. 

135,000 

406 

+  + 

+     o 

15.  . 

230,000 

21 

O        0 

0        O 

16.  . 

250,000 

37 

+     o 

0        O 

!8.. 

110,000 

40 

0        0 

+     o 

20.  . 

90,000 

54 

+     o 

0        0 

21.  . 

220,000 

22 

0        O 

0        0 

23.  . 

+     o 

0        0 

Average  .... 

240,000 

86 

33% 

22% 

Average  removal  99.96  per  cent.         99-993  per  cent. 


198  Elements  of  Water  Bacteriology. 

The  success  of  chemical  disinfection  varies  with  the 
character  of  the  sewage  or  effluent  treated  since  -the 
organic  matter  present  consumes  a  certain  amount  of 
the  disinfectant  and  renders  it  inoperative.  Discordant 
results  are  therefore  reported  from  different  sources. 

An  important  series  of  experiments  carried  out  in  Ohio 
by  Kellerman,  Pratt,  and  Kimberly  (1907)  showed  good 
results  with  sand  filter  effluents  and  contact  Affluents. 
Septic  sewage,  on  the  other  hand,  required  large  amounts 
of  chlorine  to  produce  a  reasonable  bacterial  reduction. 
The  following  table,  on  page  199,  shows  the  results 
obtained  at  Marion,  Ohio. 

In  Germany,  on  the  other  hand,  Schumacher  (1905), 
Kranepuhl  (1907),  and  Kurpjuweit  (1907)  found  larger 
amounts  of  chlorine  necessary,  in  the  neighborhood  of  60 
parts  per  million  parts  of  sewage.  Their  tests  were  some- 
what severe,  however,  the  criterion  of  success  being  the 
absence  of  B.  coli  in  a  large  proportion  of  liter  samples. 

The  science  of  sewage  bacteriology  is  in  its  infancy; 
and  it  is  difficult  to  give  any  general  rules  for  the  inter- 
pretation of  bacteriological  examinations  designed  to  in- 
dicate whether  disposal  plants  are  successful  or  not. 
Houston  stated  provisionally  that  the  20°  count  should 
be  under  100,000,  and  the  37°  count  under  10,000, 
while  B.  coli  should  be  absent  from  .001  c.c.  and  B. 
sporogenes  from  .1  c.c.  (Houston,  i9O2b).  This  standard 
seems  to  us  far  too  lenient.  Either  organic  purity  alone 
is  necessary,  as  at  many  sewage  disposal  plants,  or  a 


Sewage  and  Sewage  Effluents. 


199 


BACTERIA  IN  SEPTIC  EFFLUENT,  CONTACT  EFFLUENT, 
AND  SAND  EFFLUENT  AT  MARION,  O.,  BEFORE  AND 
AFTER  TREATMENT  WITH  CALCIUM  HYPOCHLORITE. 

(KELLERMAN,  PRATT,  AND  KIMBERLY,   1907.) 


Bacteria  per  c.c. 

Average 

Available 

Date. 

Effluent. 

Chlorine. 

20°  C. 

37°  C.    Total  Count. 

Parts  per 

Million. 

Untreated. 

Treated. 

Untreated. 

Treated. 

1907. 
Apr.  ii 

Septic 

4-3 

850,000 

1,100,000 

1,200,000 

240,000 

Apr.  12 

Septic 

6.2 

4,400,000 

550,000 

850,000 

260,000 

Apr.  15 

Septic- 

7-6 

600,000 

400,000 

450,000 

190,000 

Apr.  28 

Contact 

2.9 

110,000 

2,500 

Apr.  29 

Contact 

5-° 

65,000 

1,  600 

73,000 

37° 

Apr.    3 

Contact 

4-4 

500,000 

800 

160,000 

400 

Mar.  21 

Sand 

3-8 

49,000 

570 

9,800 

i5° 

Mar.  22 

Sand 

3-° 

56,000 

140 

7,000 

60 

Mar.  26 

Sand 

i-5 

70,000 

4,000 

20,000 

1  60 

Average 

Bacteria  per  c.c. 

Available 

Date. 

Effluent. 

Chlorine  . 

Parts  per 

37°  C,  Red  Colonies. 

B.  Coli. 

Million. 

Untreated. 

Treated. 

Untreated. 

Treated. 

1907. 

Apr.  ii 

Septic 

4-3 

55>000 

7,400 

Apr.  12 

Septic 

6,2 

60,000 

15,000 

Apr.  15 

Septic 

7.6 

100,000 

51,000 

Apr.  28 

Contact 

2.9 

20,000 

Not  in   .  c 

Apr.  29 

Contact 

5-° 

10,000 

o 

15,000 

"     "     -5 

Apr.    3 

Contact 

4.4 

21,000 

3 

20,000 

"     "  i.o 

Mar.  21 

Sand 

3-8 

1,300 

0 

I,OOO 

"     "  i.o 

Mar.  22 

Sa-nd 

3.0 

800 

0 

2,000 

"     "i.o 

Maif.  26 

Sand 

i-5 

4,000 

I 

2,000 

In  i.o 

2OO  Elements  of  Water  Bacteriology. 

higher  grade  of  purity  than  this  should  be  attained.  It 
seems  wisest  at  the  present  time  to  avoid  fixing  any  gen- 
eral standards  of  purity  for  sewage  effluents.  Each  case 
should  be  judged  intelligently  on  its  own  merits.  In 
general,  however,  where  bacterial  purification  is  indi- 
cated at  all,  it  seems  fair  to  demand  that  the  effluent 
should  be  of  such  a  quality  as  not  to  increase  materially 
the  bacterial  content  of  the  body  of  water  into  which  it 
is  discharged. 

Before  leaving  the  subject  of  sewage  bacteriology, 
brief  reference  must  be  made  to  the  importance  of  bacte- 
riological studies  in  relation  to  the  processes  of  sewage 
purification  which  bring  about  the  removal  of  the  organic 
matter  itself.  Nothing  is  more  necessary  to  the  develop- 
ment of  the  present  art  of  sewage  disposal  than  knowledge 
of  the  micro-organisms  concerned  and  of  the  conditions 
which  favor  their  activity;  but  such  knowledge  is  woe- 
fully deficient.  Something  is  known  of  the  nitrifying 
organisms  long  ago  discovered  by  Winogradsky.  Such 
recent  work  as  that  of  Schultz-Schultzenstein  (1903), 
Boullanger  and  Massol  (1903),  and  Calmette  (1905),  has 
cleared  up  many  points  concerning  these  forms;  but 
much  remains  to  be  done.  In  regard  to  the  reducing 
action  of  bacteria  in  the  septic  tank  and  contact  bed  we 
are  almost  wholly  in  the  dark.  Septic  tanks  work  well 
with  some  sewages  and  badly  with  others;  and  the  pres- 
ence or  absence  of  the  right  bacteria  is  probably  largely 
responsible  for  the  different  results.  In  some  cases,  as  at 


Sewage  and  Sewage  Effluents.  201 

Plainfield,  N.  J.,  the  seeding  of  a  tank  with  cesspool 
contents  has  produced  a  material  improvement  in  septic 
action. 

Knowledge  of  the  kinds  of  bacteria  involved  would 
make  it  possible  to  substitute  scientific  control  for  such 
empiricism  and  might  well  lead  to  improved  methods  of 
a  more  intensive  character  than  are  yet  available.  The 
work  already  done  upon  a  laboratory  scale  furnishes 
promise  of  such  results.  The  student  who  wishes  to 
follow  out  this  line  of  investigation  will  find  a  good  sum- 
mary of  what  is  already  known  of  the  hydrolysis  and 
denitrification  of  nitrogenous  bodies  and  the  decomposi- 
tion of  cellulose  and  other  carbohydrates  in  Rideal's  "  Sew- 
age, and  the  Bacterial  Purification  of  Sewage  "  (1906). 

Gage  (1905)  has  made  a  suggestive  study  of  the  bac- 
teria which  carry  on  the  reducing  changes  in  sewage 
which  deserves  the  study  of  all  who  are  interested  in  the 
more  theoretical  aspects  of  sewage  treatment.  His  method 
consisted  in  plating  sewages  and  effluents,  isolating  typi- 
cal cultures  and  determining  their  power  to  decompose 
peptone  and  nitrates  with  the  production  of  ammonia 
and  free  nitrogen.  The  rate  of  gelatin  liquefaction,  the 
amount  of  nitrate  reduced,  the  amount  of  free  ammonia 
formed,  and  the  amount  of  nitrogen  liberated  were  quan- 
titatively determined  for  each  culture  thus  isolated. 

The  numerical  values  obtained,  multiplied  by  the  num- 
ber of  bacteria,  apparently  of  the  same  type,  observed  in 
the  plates,  gave  coefficients  of  the  liquefying,  denitrifying, 


2O2  Elements  of  Water  Bacteriology. 

ammonifying,  and  nitrogen  liberating  power  of  the  efflu- 
ent; and  these  coefficients  may  be  considered  as  measures, 
for  a  given  sample,  of  the  tendency  of  the  bacterial 
flora  to  set  up  certain  changes.  The  results  of  further 
studies  made  by  Clark  and  Gage  (1905),  on  sewages  and 
on  sand,  contact,  and  trickling  effluents,  show  that  there 
may  be  important  differences  between  various  sewages  in 
this  respect  which  must  render  their  purification  more 
or  less  easy.  They  indicate  that  the  effluents  obtained 
from  intermittent  sand  filters  in  cold  weather  contain 
larger  numbers  of  ammonifying  and  denitrifying  bacteria 
than  appear  at  other  seasons,  which  may  help  to  explain 
the  poorly  nitrified  effluents  obtained  in  the  winter  season. 
Along  these  and  similar  lines  research  work  in  sewage 
bacteriology  promises  to  be  fruitful  of  results. 


APPENDIX. 


MEDIA   MAKING. 

Extract  from  the  Report  of  the  Committee  on  Standard  Methods  of 
Water  Analysis,  1905. 

IN  view  of  the  marked  influence  upon  bacteriological 
reactions  of  variations  in  culture  media  caused  by  differ- 
ences both  in  ingredients  and  in  technique  of  prepara- 
tion, it  is  necessary  that  uniform  methods  be  used  in  order 
to  obtain  comparable  data.  In  specifying  the  various  in- 
gredients used  in  culture  media  it  is  the  intention  of  the 
committee  that  they  shall  be  uniform  in  quality,  but  it 
is  not  the  intention  that  the  recommendations  as  to  ingre- 
dients and  technical  manipulations  shall  stand  in  the 
way  of  true  progress  as  to  improvements.  When,  how- 
ever, improved  or  modified  methods  are  used,  the  varia- 
tions from  the  standard  methods  shall  be  plainly  set 
forth  together  with  the  reasons  for  the  modifications. 

INGREDIENTS. 

Distilled  water  shall  be  used  in  the  preparation  of 
standard  culture  media. 

Infusions  of  fresh  lean  meat,  and  not  meat  extract, 
shall  be  used  as  the  basis  of  various  media. 

203 


2O4  Appendix. 

Sodium  chloride  shall  not  be  added  to  any  culture 
medium  herein  specified. 

Pepton  shall  be  that  of  Witte  (dry  from  meat). 

Gelatin  shall  be  the  best  French  brand,  so-called.  It 
shall  be  as  free  as  possible  from  acids  and  other  impuri- 
ties, and  shall  be  of  such  a  character  that  a  10  per  cent 
solution  prepared  in  the  usual  way  shall  not  soften  when 
kept  at  a  temperature  of  25°  C. 

Commercial  agar  in  threads  shall  be  of  as  high  a 
grade  as  can  be  obtained.  Agar  may  be  purified  by 
washing. 

The  various  sugars,  such  as  dextrose,  lactose,  and  sac- 
charose, shall  be  as  nearly  as  possible  the  chemically 
pure  compounds  designated.  Unusual  effort  to  obtain 
such  sugars  is  considered  to  be  necessary. 

Glycerine  shall  be  double  distilled. 

In  place  of  litmus,  azolitmin*  shall  be  used  as  a  i  per 
cent  aqueous  solution. 

Of  the  various  other  ingredients  used,  nearly  all  of 
which  are  of  a  mineral  nature,  special  effort  shall  be 
made  to  see  that  they  are  chemically  pure  products  within 
the  full  meaning  of  this  expression. 

STERILIZATION. 

Of  the  two  available  methods  of  sterilization,  the  inter- 
mittent method  at  a  temperature  of  100°  C.  is  considered 
on  the  whole  to  be  preferable.  The  higher  temperatures 

*  Preferably  Kahlbaum's  (authors). 


Appendix.  205 

of  the  autoclave  facilitate  chemical  reactions  and  changes 
which  in  some  cases  are  undesirable. 

When  the  latter  method  is  used,  media  contained  in 
ordinary  receptacles  shall  be  sterilized  by  exposure  in  an 
autoclave  at  a  temperature  of  120°  C.  (15  pounds  pres- 
sure) for  five  minutes.  Where  media  are  sterilized  in 
large  bulk,  the  period  of  heating  shall  be  extended  to  12 
minutes.  It  is  preferable,  however,  to  sterilize  media  in 
reasonably  small  containers  (500  to  700  c.c.). 

In  intermittent  sterilization,  media  shall  be  placed  on 
each  of  three  successive  days  in  streaming  steam  for  30 
minntes  after  the  steam  fills  the  sterilizer. 

REACTION. 

Phenolphthalein  shall  be  the  standard  indicator  used 
in  obtaining  the  reaction  of  all  media.  Turmeric  paper 
possesses  similar  properties,  and  its  use  is  advised  where 
phenolphthalein  is  not  available. 

Titrations  and  adjustment  of  reactions  shall  be  made 
as  follows: 

Put  5  c.c.  of  the  medium  to  be  tested  into  45  c.c. 
distilled  water.  Boil  briskly  one  minute.  Add  i  c.c. 
of  phenolphthalein  solution  (5  g.  of  commercial  salt  in 
one  liter  of  50  per  cent  alcohol.)  Titrate  while  hot 

N 

(preferably  while  boiling)  with  —  caustic  soda.     A  faint 

20 

but  distinct  pink  color  marks  the  true  end-point.  This 
distinct  pink  color  may  be  more  precisely  described  as  a 


206  Appendix. 

combination  of  25  per  cent  of  red  (wave  length  approxi- 
mately 658)  with  75  per  cent  of  white  as  shown  by  the 
disks  of  the  color  top,  described  under  Record  of  Tints 
and  Shades  of  Apparent  Color.  In  practice  titration 
is  continued  until  the  pink  color  of  alkaline  phenol- 
phthalein  matches  that  of  the  fused  disks. 

All  reactions  shall  be  expressed  with  reference  to  the 
phenolphthalein  neutral  point  and  shall  be  stated  in  per- 
centages of  normal  acid  or  alkaline  solutions  required  to 
neutralize  them.  Alkaline  media  shall  be  recorded  with 
the  minus  ( — )  sign  before  the  percentage  of  normal  acid 
needed  for  their  neutralization,  and  acid  media  with  the 
plus  (+)  sign  before  the  percentage  of  normal  alkaline 
solution  necessary  for  their  neutralization. 

The  standard  reaction  of  culture  media  shall  be  +  i.o 
per  cent.  If  it  differs  from  i  per  cent  by  more  than  0.2 
per  cent  it  should  be  readjusted. 

Wherever  reactions  other  than  the  standard  above 
given  are  used  it  shall  be  clearly  stated  in  all  results  of 
bacterial  work,  and  the  reasons  therefor  also  stated. 

STORAGE  OF  MEDIA. 

It  is  recognized  by  the  committee  that  it  is  desirable 
to  prepare  media  in  large  quantities  in  order  to  guard 
against  discrepancies  in  composition;  but,  all  things  con- 
sidered, the  complications  .resulting  from  the  varying 
amounts  of  heating  incident  to  withdrawing  portions 
from  time  to  time  and  tubing  it,  are  believed  to  more 


Appendix.  207 

than  offset  this  advantage.  Consequently,  when  possible, 
media  shall  be  put  at  once  into  tubes  and  placed  in  cold 
storage. 

To  guard  against  changes  due  to  evaporation  all  media 
not  used  promptly  shall  be  stored  in  a  moist  atmosphere, 
preferably  in  an  ice-box,  or  else  the  flask  shall  be  sealed 
by  dipping  the  cotton  plug  in  paraffin. 

NUTRIENT  BROTH. 

Nutrient  broth  shall  be  prepared  as  follows:  Infuse 
500  g.  chopped  lean  meat  24  hours  with  1000  c.c.  dis- 
tilled water,  in  refrigerator.  Restore  loss  by  evaporation. 
Strain  infusion  through  cotton  flannel.  Add  one  per  cent 
pepton.  Warm  on  water  bath,  stirring  until  the  pep- 
ton  is  dissolved.  Heat  over  boiling  water  (or  steam) 
bath  30  minutes.  Restore  loss  by  evaporation.  Titrate. 
Adjust  reaction  to  +  i  per  cent  by  adding  normal  hydro- 
chloric acid  or  normal  sodium  hydrate,  as  required.  Boil 
two  minutes  over  free  flame,  constantly  stirring.  Restore 
loss  by  evaporation.  Filter  through  absorbent  cotton  and 
cotton  flannel,  passing  the  liquid  through  until  clear. 
Titrate  and  record  final  reaction.  Tube,  using  10  c.c.  in 
each  tube.  Sterilize. 

SUGAR   BROTHS. 

Sugar  broths  shall  be  prepared  in  the  same  general 
manner  as  the  standard  nutrient  broth,  with  the  addition 
of  one  per  cent  of  dextrose,  lactose,  saccharose  or  other 
sugar;  or  the  sugar  may  be  added  to  the  completed 


208  Appendix. 

nutrient  broth  just  before  sterilizing.  Except  in  the  case 
of  dextrose  broth  it  is  important  that  the  muscle-sugar  in 
the  meat  infusion  be  removed  by  inoculating  with  B.  coli. 

The  reaction  of  sugar  broths  shall  be  neutral  to  phenol- 
phthalein. 

Sterilization  shall  be  done  in  streaming  steam  in  the  case 
of  all  sugar  broths  to  prevent  inversion  of  the  sugar. 

For  the  routine  work  of  testing  samples  of  water  for 
B.  coli,  especially  large  volumes  of  water  are  to  be  mixed 
with  broths  of  such  strength  as  to  make  the  resulting 
mixture  one  of  normal  strength.  Liebig's  Beef  Extract 
may  be  substituted  for  beef  infusion  in  the  preparation 
of  dextrose  broth  only:  three  grams  of  the  beef  extract 
for  each  liter  of  broth. 

NUTRIENT  GELATIN  AND  AGAR. 

Nutrient  gelatin  and  agar  shall  be  prepared  as  follows : 

Gelatin.  Agar. 

1.  Boil  15  g.  thread  agar  in  500  c.c. 

water  for  half  an  hour  and  make  up 
weight  to  500  g.  or  digest  for  10 
minutes  in  the  autoclave  at  110°  C. 
Let  this  cool  to  about  60°  C. 

2.  Infuse  500  g.   lean  meat  24       Infuse  500  g.  lean  meat  24  hours 
hours  with  1000  c.c.  of  dis-       with  500  c.c.  of  distilled  water  in 
tilled  water  in  refrigerator.  refrigerator. 

3.  Make  up  any  loss  by  evaporation. 

4.  Strain  infusion  through  cotton  flannel. 

5.  Weigh  filtered  infusion. 

6.  Add  one  per  cent  Witte's  pep-       Add  two  per  cent  of  Witte's  pep- 
ton    and    10   per  cent    gold       ton. 

label  sheet  gelatin. 


Appendix.  209 

Gelatin.  Agar. 

7.  Warm  on  water  bath,  stirring  till  pepton  and 
gelatin  are  dissolved  and  not  allowing  the 
temperature  to  rise  above  60°  C. 

8.  To  500  g.  of  the  meat  infusion  add 

500  c.c.  of  the  three  per  cent  agar, 
keeping    the    temperature    below 
60°  C. 

9.  Heat  over  boiling  water  (or  steam)  bath  for 
30  minutes. 

10.  Restore  loss 'by  evaporation. 

11.  Titrate,  after  boiling  one  minute  to  expel  car- 
bonic acid. 

12.  Adjust  reaction  to  +  i.o  per  cent  by  adding 
normal  hydrochloric  acid  or  sodium  hydrate 
as  required. 

13.  Boil  two  minutes  over  free  flame,  constantly 
stirring. 

14.  Make  up  loss  by  evaporation. 

15.  Filter  through  absorbent  cotton  and  cotton 
flannel,  passing  the  nitrate  through  the  filter 
until  clear. 

1 6.  Titrate  and  record  the  final  reaction. 

17.  Tube,  using  10  c.c.  of  medium  in  each  tube. 

1 8.  Sterilize  five  minutes  in  the  autoclave  at  120 
degrees,  or  for  30  minutes  in  streaming  steam 
on  three  successive  days.     Put  the  gelatin  at 
once  into  ice-water  till  solidified. 

19.  Store  in  the  ice-chest  in  a  moist  atmosphere, 
to  prevent  evaporation. 

LACTOSE  (OR  DEXTROSE)  LITMUS  AGAR. 

Lactose  or  dextrose  litmus  agar  shall  be  prepared  in  the 
same  manner  as  nutrient  agar,  with  the  addition  of  one 
per  cent  of  lactose  (or  dextrose)  to  the  medium  just  before 
sterilization.  The  reaction  shall  be  made  neutral  to 
phenolphthalein. 


2io  Appendix. 

If  the  medium  is  to  be  used  in  tubes  the  sterilized 
azolitmin  solution  shall  not  be  added  until  just  before  the 
final  sterilization. 

If  the  medium  is  to  be  used  in  Petri  dishes  the  steril- 
ized azolitmin  solution  shall  not  be  added  to  the  medium 
until  it  is  ready  to  be  poured  into  the  dishes. 

MILK. 

The  milk  to  be  used  as  a  culture  medium  shall  be  as 
fresh  as  possible,  "Certified  Milk"  being  ordinarily  the 
best  obtainable  in  city  laboratories.  It  shall  be  placed  in 
a  refrigerator  over  night  to  allow  the  cream  to  rise  and 
the  suspended  matter  to  settle.  The  skimmed  milk  shall 
be  siphoned  off  into  a  flask  for  use.  It  will  be  found 
more  convenient,  however,  to  allow  the  milk  to  stand  in 
a  separating  funnel.  Should  the  milk  be  too  acid  the 
reaction  shall  be  corrected  to  +  i  per  cent  by  the  addition 
of  normal  sodium  hydrate.  It  is  then  ready  to  be  tubed 
and  sterilized.  Litmus  milk  shall  be  prepared  as  above, 
with  the  addition  of  sterile  i  per  cent  azolitmin.  As  it 
is  impossible  to  make  each  lot  of  litmus  milk  with  the 
same  shade  of  color,  it  is  recommended  that  a  control 
tube  be  always  exposed  with  the  inoculated  tubes  for 
purposes  of  comparison. 

NITRATE  BROTH. 

Dissolve  one  gram  pepton  in  one  liter  of  tap  water, 
and  add  0.2  grams  of  nitrite-free  potassium  nitrate. 
It  is  convenient  to  prepare  a  stock  solution  of  potas- 


Appendix.  211 

sium  nitrate  by  dissolving  four  grams  of  solid  nitrate  in 
100  c.c.  of  distilled  water  and  use  five  c.c.  of  this  solution 
in  the  above  formula.  Ten  c.c.  of  the  medium  thus 
prepared  shall  be  placed  in  a  test  tube  and  sterilized  in 
the  usual  way. 

BROTH   FOR  INDOL  TEST. 

Standard  broth  may  be  used  for  the  indol  test  if  pre- 
cautions are  taken  to  remove  the  muscle  sugar,  by  inocu- 
lating the  beef  infusion  with  B.  coli  before  making  the 
broth. 

Pepton  solution  (one  per  cent  pepton  in  water),  how- 
ever, is  preferred  by  some  for  use  in  the  indol  test,  and 
is  considered  generally  to  be  satisfactory.  Sodium  nitrite 
(o.oi  per  cent)  shall  be  added  in  all  cases. 

APPARATUS. 

Few  definite  requirements  need  be  made  respecting 
apparatus.  The  quality  of  the  glass  shall  be  such  as  not 
to  be  easily  acted  upon  by  the  reagents  used,  and  all 
glassware  shall  be  scrupulously  clean  when  used.  When 
necessary  it  shall  be  sterilized  by  dry  heat  for  one  hour 
at  about  150°  C.  A  slight  browning  of  the  cotton  stop- 
pers is  a  good  index  of  proper  exposure. 

In  some  operations,  as,  for  example,  the  determination 
of  the  thermal  death  point,  it  is  necessary  to  use  test 
tubes  of  a  definite  size  and  thickness.  For  this  purpose 
the  standard  size  culture  tube  shall  be  15  cm.  long,  1.6  cm. 


212          .  Appendix. 

in  diameter,  and  of  medium  weight.  Tubes  to  be  filled 
with  gelatin  for  quantitative  work  may  be  those  described 
as  6  in.  X  f  in.  "heavy." 

The  standard  loop  for  making  transfers  shall  be  pre- 
pared as  follows: 

Bend  the  end  of  a  piece  of  No.  27  platinum  wire  about 
10  cm.  long  over  a  bit  of  No.  10  wire,  and  fasten  the  loop 
thus  formed  into  a  glass  rod  to  serve  as  a  handle.  A 
loopful  of  culture  shall  be  interpreted  as  meaning  all  the 
fluid  that  the  loop  can  hold.  That  is,  the  fluid  shall 
form  a  bi-convex  body  and  shall  not  be  simply  a  film 
covering  the  space  in  the  loop. 

The  standard  fermentation  tube  shall  be  a  tube  1.5  cm. 
in  diameter,  bent  at  an  acute  angle,  closed  at  one  end 
and  provided  with  a  bulb  at  the  other  which  is  large 
enough  to  receive  all  the  liquid  contained  in  the  closed 
portion.  The  length  of  the  closed  end  of  the  tube  shall 
be  about  8  cm. 

INCUBATION. 

There  shall  be  two  standard  temperatures  of  incuba- 
tion for  special  work,  namely,  20°  C.  and  37°  C.,  the 
first  corresponding  to  ordinary  room  temperature,  the 
second  to  blood  heat.  The  temperature  of  the  incu- 
bators shall  not  be  allowed  to  vary  from  these  two  stand- 
ards by  more  than  i°  C.  in  either  direction. 

The  atmosphere  of  the  incubator  shall  be  kept  moist, 
preferably  near  the  point  of  saturation.  The  incubator 


Appendix.  213 

shall  be  ventilated  so  as  to  insure  a  reasonably  good  cir- 
culation of  air  in  order  to  prevent  the  accumulation  in 
the  incubator  of  gases  which  might  be  prejudicial  to  the 
development  of  the  bacteria. 

No  definite  period  of  incubation  can  be  prescribed 
which  will  be  suitable  for  all  the  work  of  species  deter- 
mination, but  in  reporting  results  the  period  used  shall 
always  be  stated  and  form  a  part  of  the  report.  General 
statements  as  to  the  necessary  periods  will  be  found  in 
connection  with  the  principal  tests. 

PRELIMINARY  CULTIVATION. 

It  is  impossible  to  control  completely  the  original 
vitality  of  bacteria  when  ready  for  cultivation,  because 
in  most  cases  the  conditions  for  their  optimum  growth 
are  not  known.  Experience,  however,  has  shown  that, 
when  bacteria  are  submitted  to  a  period  of  preliminary 
cultivation  or  rejuvenation  in  nutrient  broth  and  trans- 
fers of  young  cultures  made  from  one  tube  to  another  at 
frequent  intervals,  the  result  is  to  put  the  bacteria  into  a 
condition  where  subsequent  cultures  give  greater  uni- 
formity in  their  characteristics  than  where  this  procedure 
is  not  followed.  The  following  shall  be  considered  as 
the  standard  procedure  for  this  preliminary  cultivation, 
and  all  bacteria  shall  be  so  treated  before  proceeding  to 
the  detailed  tests. 

Procedure.  —  Make  a  transfer  from  an  agar  culture  of 
the  bacterium  to  be  tested  into  a  tube  of  nutrient  broth, 


214  Appendix. 

and  incubate  for  24  hours  at  20°  C.  Transfer  from  this 
culture  to  a  second  tube  of  broth,  and  again  incubate  for 
24  hours  at  20°  C.  Transfer  from  this  second  culture  to 
a  third  tube  of  broth  and  incubate  again  for  24  hours  at 
20°  C.  Frorh  this  third  broth  culture  make  a  gelatin 
plate  and  incubate  for  48  hours  at  20°  C.  (This  is  to 
prevent  working  with  a  possible  mixed  culture  due  to 
accidental  contamination.)  From  one  of  the  colonies  on 
the  gelatin  plate  transfer  to  a  tube  of  slanted  agar,  incu- 
bate at  20  degrees  for  48  hours,  and  use  this  culture  for 
making  subsequent  inoculations  in  the  various  media. 

ADDITIONAL   FORMULAE. 

LOEFFLER'S  BLOOD  SERUM. 

This  medium  consists  of  3  parts  blood  serum  and  i  part 
of  i  per  cent  broth  with  reaction  +  0.8.  The  serum 
is  obtained  from  fresh  beeves'  blood,  which  is  collected 
in  sterile  jars  and  allowed  to  stand  for  24  hours  in  the 
/refrigerator  for  coagulation.  The  serum  is  then  drawn 
off,  filtered,  and  mixed  with  the  dextrose  broth  in  the 
proportion  above  indicated.  Hill  finds  that  filtering  the 
serum  through  the  coagulum  obtained  after  adjusting  the 
reaction  of  the  broth,  gives  a  filtrate  which  is  clear  and 
almost  colorless  (Hill,  1899). 

Tubes  are  filled  with  the  mixture,  placed  in  trays  so 
that  the  desired  slant  is  obtained,  and  carefully  heated 
in  a  Koch  coagulator  containing  cold  water  in  the  water- 
jacket.  This  water  is  brought  to  a  boil  and  kept  boiling 


Appendix.  215 

for  three  hours.  Repeating  this  process  on  three  suc- 
cessive days  solidifies  the  serum  so  that  it  may  be  subse- 
quently sterilized  in  flowing  stream  for  twenty  minutes 
on  three  successive  days. 

PHENOL   BROTH. 

To  1000  c.c.  water  add  separately,  and  in  the  following 
order,  100  grams  dextrose,  50  grams  pepton,  i.o  grams 
phenol.  Heat  until  all  constituents  are  dissolved,  boil 
for  fifteen  minutes,  and  sterilize  for  fifteen  minutes  at 

IIO-I20°C. 

NEUTRAL  RED   BROTH. 

To  neutral  broth  is  added  0.5  per  cent  dextrose  and 
i  per  cent  of  a  0.5  per  cent  aqueous  solution  of  Griibler's 
neutral  red.  Sterilize  at  100°. 

MACCONKEY'S  MEDIA. 

A.  Agar. 

Agar 1.5  grams 

Sodium  taurocholate  (pure)      0.5  gram 

Pepton 2.0  grams 

Water 100.0  c.c. 

This  is  boiled,  clarified,  and  filtered  as  usual,  then 
i.o  gram  lactose  is  added,  and  the  medium  tubed  and 
sterilized  for  three  successive  days  at  100°. 

B.  Broth. 

Sodium  taurocholate  (pure) 0.5  gram 

Pepton 2.0  grams 

Glucose 0.5  gram 

Water 100.0  grams 

Boil,  filter,  and  add  sufficient  neutral  litmus,  fill  fer- 
mentation tubes,  and  sterilize  at  100  degrees. 


2 1 6  Appendix. 

Litmus  Solution.  —  To  one-half  pound  of  litmus  cubes 
add  enough  water  to  more  than  cover;  boil,  and  decant 
off  the  solution.  Repeat  this  operation  with  successive 
small  quantities  of  water  until  from  3  to  4  liters  of  water 
have  been  used  and  the  cubes  are  well  exhausted  of  color- 
ing matter.  Pour  the  decantations  together  and  allow 
them  to  settle  over  night.  Siphon  off  the  clear  solution. 
Concentrate  to  about  i  liter  and  make  the  solution  de- 
cidedly acid  with  glacial  acetic  acid.  Boil  down  to  about 
one-half  liter  and  make  exactly  neutral  with  caustic  soda 
or  potash.  To  test  for  the  neutral  point,  place  one  drop  of 

N 

the  solution  in  a  test  tube.     One  drop  of  —    HC1  should 

20 

N 

turn  the  drop  red,  while  one  drop  of  —  NaOH  should 

20 

turn  it  blue.  Filter  the  solution  and  sterilize  at  110°  C. 
This  solution  should  be  added  to  the  media  just  before 
use  in  the  proportion  of  about  J  c.c.  to  5  c.c.  of  medium. 
For  the  methods  of  preparation  of  other  special 
culture  media  the  student  is  referred  to  the  papers 
published  by  the  investigators  who  have  originated  the 
media  in  question. 


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INDEX    OF   AUTHORS 


Abba,  116,   131. 
Abbott,  33. 
Adami,  72. 
Adams,  26,  39,  41. 
Altschuler,  74. 

American  Committee  on  Standard 
Methods,  28, 29, 66, 89,95, 97>IO4- 
Amyot,  113. 
Andrewes,  69,  153,  160. 

Baker,  154,  155,  156.  157. 

Barthel,  119. 

Baton,  93,  97,  144. 

Beckmann,  114. 

Belcher,  162. 

Belitzer,  112. 

Blachstein,  116. 

Blunt,  17. 

Bolton,  48. 

Boullanger,  200. 

Braun,  150. 

Brezina,  51. 

Brotzu,  112. 

Brown,  122. 

Bruns,  116. 

Buchner,  17. 

Bulstrode,  181. 

Burn,  121. 


Calmette,  193,  200. 

Cambier,  33,  42,  43- 

Carpenter,  197. 

Chick,  60,  89,  124. 

Chopin,  72. 

Christian,  94. 

Clark,  52,  119,  129,  132,  136,  145, 

190,  202. 
Conradi,  69. 
Copeland,  89. 

Dibdin,  184. 
Ditthorn,  76. 
Downes,  17. 
Drigalski,  69,  77. 
Drown,  171. 
Diiggeli,  119. 
Dunbar,  83. 
Dunham,  52,  83. 
Durham,  88,  163,  164. 
Dyar,  112. 

Eddy,  188. 
Egger,  22. 
Eijkman,  93. 
Ellms,  33. 
Eisner,  68. 
Endo,  70. 


247 


248 


Index. 


English   Committee,   42,   61,   66, 

92,  103,  159. 
Escherich,  86. 
Eyre,  113. 

Ferguson,  97,  144. 
Fehrs,  15. 
Ficker,  71,  75. 
Fischer,  81. 
Fischer,  A.,  2. 
Flatau,  81. 
Ford,  88. 

Frankland,  12,  33,  82,  84. 
Fremlin,  112. 
Freudenreich,  16,  121. 
Frost,  1 6,  107. 
Fuller,  C.  A.,  21,  82. 
Fuller,  G.  W.,  28,  38,  53,  97,  no, 
144. 

Gaehtgens,  70. 

Gage,  9,  25,  26,  27,  38,  39,  41,  63, 
64,  90,  94,  99,  109,  no,  119, 
129,  132,  136,  142,  144,  145, 
151,  154,  173,  175,  186,  201, 
202. 

Garre,  16. 

Gartner,  14,  48. 

Gautie,  139. 

Gildemeister,  76. 

Gordan,  119. 

Gordon,  160. 

Gotschlich,  84. 

Hammerl,  122. 
Hankin,  68. 
Hansen,  177. 
Heider,  182. 


Heraeus,  31. 

Hesse,  25,  40,  42. 

Hill,  33>  59>  146. 

Hiss,  80. 

Hoffmann.  21,  71. 

Border,  160. 

Horrocks,    16,   23,    78,    124,    154, 

158,  162. 
Horton,  132. 
Houston,  23,  113,  119,  120,  123, 

130,    131,    133,    153,    158,    160, 

162,   165,    167,    176,    178,    185, 

192,    195,    196.   198. 
Hunnewell,  95,  127,  128,  154. 
Huntemiiller,  15. 

Irons,  90,  92,  142,  151. 

Jackson,  92,  146,  147. 

Janowski,  7. 

Johnson,  53,  no,  113,  188,  191, 

192,  193. 
Jordan,  15,  16,  18,  20,  21,  36,  50, 

82,  125,  126,  182. 

Kabrhel,  22. 

Kaiser,  133. 

Keith,  112. 

Kellerman,  198,  199. 

Kershaw,  192. 

Kimberley,  198,  199. 

Kisskalt,  9. 

Klein,  68,  119,  161,  162,  176. 

Klotz,  73. 

Kloumann,  72. 

Koch,  83. 

Kohn,  35,  49. 

Kolle,  84. 


Index. 


249 


Konradi,  21. 
Korschun,  15. 
Kranepuhl,  198. 
Kruse,  114. 
Kiibler,  80. 
Kurpjuweit,  198. 
Kusel,  no. 

Laws,  69,  153. 
LeGros,  154. 
Lentz,  70. 
Levy,  1 1 6. 
Lochridge,  19. 
Loeffler,  70,  83. 
Longley,  93,  97,  144- 
Lubenau,  78. 

MacConkey,  146. 

McGowan,  192. 

McWeeny,  104. 

Makgill,  150. 

Mascbek,  23,  31. 

Massachusetts     State     Board     of 

Health,  23,  103,  132,  136. 
Massol,  200. 
Mathews,  61,  88. 
Mayer,  51,  82. 
Meyer,  18,  19. 
Miquel,  6,  33,  42.,  43,  46,  50. 
Moore,  113. 
Moroni,  116. 
Muller,  27,  75. 

Neufeld,  80. 

Neumann,  13,  94. 

Nibecker,  12,  62,  63,  99,  143,  155. 

Niedner,  25,  42. 

Nieter,  76. 


Nowack,  94. 

Nuttall,  161.  i 

Orlandi,  131. 
Osgood,  182. 
Otto,  13. 

Pakes,  124. 

Papasotiriu,  119. 

Parietti,  67. 

Park,  80. 

Pennington,  no. 

Petruschky,  138. 

Phelps,  25,  27,  38,  39,  65,  99,  109, 

145,  146,  151,  189,  194,  197. 
Philbrick,  n,  50. 
Poujol,  115. 
Pratt,  198,  199. 
Prausnitz,  50. 
Prescott,  12,  38,  90,  118,  154,  155, 

156,  157- 
Pritzkow,  193. 
Procaccini,  17. 
Pusch,  138. 

Rapp,  1 8,  20. 
Refik,  115. 
Reinsch,  38. 
Remlinger,  82. 
Reynolds,  91. 
Rideal,  61,  196. 
Riedel,  35. 
Rondelli,  131. 
Roth,  71. 
Rothberger,  150. 
Russell,  13,  16,  21,  82. 

Savage,  21,  58,  113,  117,  138,  150, 
1 68,  181. 


250 


Index. 


Sawin,  148. 
Schepilewski,  74. 
Scheurlen,  14. 
Schneider,  82. 
Schottelius,  83. 
Schiider,  75. 

Schultz-Schultzenstein,  200. 
Schumacher,  198. 
Sedgwick,  4,  17,  38,  85. 
Shuttle-worth,  55. 
Smith,  87,  90,  112,  121. 
Stamm,  105. 
Sternberg,  47. 
Stokes,  151. 
Stoughton,  144. 

Thomann,  94. 
Thomson,  68. 
Thresh,  33,  85. 
Thumm,  193. 
Tiemann,  14,  48. 
Tietz,  70. 
Tissandier,  6. 
Twort,  1 66. 


Vallet,  75. 

Vincent,  51,  139,  169,  179. 

Walker,  88,  119,  188,  190. 

Wathelet,  69. 

Weissenfeld,  117. 

Welch,  161. 

Wheeler,  18. 

Wherry,  108. 

Whipple,  9,  18,  19,  34,  35,  39,  41, 
82,  96,  101,  142,  143,  144,  177. 

Widal,  80. 

Willson,  72,  76,  77. 

Winslow,  12,  17,  19,  25,  26,  30,  55, 
61,  62,  63,  65,  88,  95,  99,  no, 
119, 127, 128, 143,  145, 146, 154, 
155,  162,  177,  188,  189,  194. 

Woodman,  177. 

Wolffliugel,  22,  35 

Wright,  113. 

Wurtz,  59. 

Zagari,  16. 
Zeit,  1 6,  21,  82. 


SUBJECT  INDEX 


Acid  forming  organisms,  60. 

in  polluted  waters,  60. 
Agar,  204,  208,  209. 
Agar  streaks,  100. 
Agglutination,  72. 
Agglutination,     effective    dilution 

for  typhoid  isolation,  73. 
Agglutination  methods,  69. 
American  sewages,  188. 
Anaerobic  bacilli,  161. 
Anaerobic  bacteria,  169. 
Animal  inoculation,  116. 
Antagonism,  16. 
Arbitrary  standards,  46,  47. 
Atmospheric  waters,  6. 
Atypical  colon  bacilli,  102,  166. 
Atypical  forms,  100. 
Atypical  gas  formation,  97. 
Atypical  gas  ratio,  142. 

Bacilli,  anaerobic,  161. 
Bacillus  alcaligenes,  163. 
Bacillus  anthracis. 

isolation  of,  from  water,  84. 
Bacillus  cloacae,  168. 
Bacillus,  coli,  86,  163. 

action  on  aldehydic  and  ketonic 
sugars,  88. 

action  on  litmus-lactose-agar,  88. 

agar  streak  of,  100. 

and   streptococci,    detection  of, 
158- 


Bacillus,  coli , —  Continued 

and    streptococci,     relation    to 
sewage  pollution,  158. 

B.   coli   and  streptococci,  relative 
growth  of,  in  dextrose  broth,  156, 

157- 
as  criterion  of  self-purification, 

122. 

as  index  of  B.  typhi,  136. 
as  index  of   sewage    pollution, 

124. 
as  measure  of,  fecal   pollution, 

139- 
as  test  for  fecal  contamination, 

175- 

as  test  organism  for  filters,  134. 
confirmatory  tests  of,  107. 
diagnostic  characters  of,  103. 
distribution,  112. 
fermentative  power  of,  87. 
gas  formation  by,  90. 
gas  ratio  of,  105,  141. 
importance  of  number  in  water, 

i38>  139- 

importance  of  number  of,  120. 

in  animals,  112,  113. 

in  bottled  waters,  132. 

incubation  of  at  high  tempera- 
tures, 94. 

in  Chicago  drainage  canal,  125. 

in  ground  waters,  131. 

in  Harrisburg  filter  effluent,  135. 


251 


252 


Subject  Index. 


Bacillus  coli,  —  Continued 
in  lakes  and  streams,  130. 
in  Lawrence  filter  effluent,  134. 
in  Merrimac  River,  134. 
in  nature,  114. 
in  polluted  waters,  124,  128. 
in  Potomac  River,  135. 
in  river  waters,  126. 
in  sewage,  185. 
in  soils,  123. 
in  spring  waters,  132. 
in  surface  waters,  130. 
in  Susquehanna  River,  135. 
in  Thames,  131, 
in  unpolluted  waters,  124,  128. 
in    Washington    filter    effluent, 

135- 

in  water  supplies,  115,  117. 

in  wells,  132,  133. 

isolation  of,  98. 

isolation  of,  by    litmus-lactose- 
agar,  97. 

mo tili ty  of,  104. 

on  plants,  118. 

overgrowth  by  streptococci,  129. 

pathogenicity  of,  116,  117. 

preliminary  enrichment  for,  89. 

presumptive  tests  for,  141. 

ratio  of,  to  total  bacteria  in  sur- 
face waters,  129. 

removal  in  pipes  and  reservoirs, 
136. 

significance  of,  112. 

typical,  i pi. 

variation  in  behavior,  no. 
B.  communior,  88. 

dysenteriae,  80,  163. 

reactions  of,  80. 

enteritidis,  163. 

enteritidis  sporogenes,  161. 


Bacillus  mycoides  group,  99. 
Bacillus  sporogenes,  161. 

characteristics  of,  162. 

occurrence  in  sewage,  162,  185. 

insolation  of,  161. 

subtilis,  1 68. 
Bacillus  typhi,  163. 

comparison  with  B.  coli,  78. 

positive  isolations  from    water, 

80,  81. 

Bacteria,  as   agents   of  decompo- 
sition, 3. 

as  agents  of  purification,  184. 

counts  of,  at  different  tempera- 
tures, 173. 

distribution,  i. 

escaping  detection,  25. 

factors  in  distribution,  i. 

food  requirements,  2. 

in  Boston  Harbor,  182. 

in  contact  beds,  193. 

in  dust  in  air,  6. 

in  effluents,  193,  194,  195. 

in  effluents  from  sewage  farms, 
192. 

in  humus,  3. 

in  ocean,  14. 

in  rain  and  snow,  6. 

in  sandfilter  effluents,  190. 

in  sea  water,  13. 

in  septic  effluents,  190. 

in  sewage,  193. 

in  soil,  3. 

in  water,  5. 

in  sand  filter  effluents,  190,  191. 

in  wells,  52. 

in  well  water,  23,  24. 

metatrophic,  2. 

microscopic  enumeration  of,  25. 

number  in  lakes  and  ponds,   12. 


Subject  Index. 


253 


Bacteria  —  Continued. 
on  the  hands,  30. 
paratrophic,  2. 
prototrophic,  2,  25. 
seasonal  variation,  7,  8. 
significance  of  high  numbers  of, 

173- 

subsidence  of,  14. 
Bacterial  analyses,  value  of,  54. 

correct   relation   to   contamina- 
tion, 9. 

correct  relation  to  stream  flow,  9. 

examination,  significance  of,  170. 
Bacteriological    examination,    ap- 
plicability of,  170. 

examination,  certainty  of,  179. 

examination,  delicacy  of,  176. 

examination,  directness  of,  1 76. 
Bile,  as  enrichment  medium,  76. 
Bile-salt  broth,  92. 
Bile  salts,  146. 
Bleaching  powder,  107. 
Body  temperature  count,  57,  173. 
Body   temperature   count,   signifi- 
cance of,  57. 
Broth,  207. 

neutral  red,  215. 

sugar,  207. 

Caffeine  fuchsin  agar,  70. 

inhibitory  action  of,  71. 

nutrose  medium,  71. 
Calcium  hypochlorite,  198. 
Chemical  disinfection,  198. 

examination  of  water,  171. 

precipitation  for  typhoid  isola- 
tion, 74,  75. 

Cholera  bacillus,  isolation  of,  from 
water,  83. 

Koch's  method,  83. 


Cholera  red  reaction,  83. 
Chromogenic  organisms  in  wells, 

24. 

Cold,  effect  of,  17. 
Cold,  effect  of,  on  typhoid  bacilli, 

ty 

Collodion  sacs,  16. 

Colon  bacilli,  atypical,  102. 

bacilli  on  plants,  118. 

bacillus,  86,  101. 

colonies  on  litmus-lactose-agar, 

99- 

diagnostic  characters  of,  103. 
distribution  of,  112. 
gas  ratio  of,  105. 
growth     on     Drigalski-Conradi 

medium,  70. 
isolation  of,  86,  98. 
group,  165. 

group,  fermentative  power  of,  87. 
group,  sub-types,  87. 
Colon -like  organisms,  102. 
test,  dilutions  for,  95. 
test  in  large  samples,  96. 
test,  24-hour  incubation,  97. 
test,  use  of  i  c.c.  samples,  96. 
typhoid  group,  163. 
Comparative    growths,    Nahrstoff 

•  agar  and  peptone  agar,  27. 
Confirmatory    tests     for    B.    coli, 

107. 
Comparison  of    sand    filters  and 

mechanical  filters,  54. 
Composition  as  effected  by  sterili- 
zation, 39. 

of  medium,  effect  of,  39. 
of  water,  effect  of,  40. 
Composition  of  glass,  importance 

of,  40. 
Concentration  methods,  69. 


254 


Subject  Index. 


Contact  beds,  192. 

Counting,  experimental   error  in, 

43>  44- 

Counting,  procedure,  42. 
Counts  at  different  temperatures, 

63- 
Counts  at  20°,  30°,  40°,  and  50°, 

173- 
Cycle  of  nitrogen,  4,  5. 

Deep  wells,  bacteria  in,  23,  24. 
Dextrose  broth  as  test  for  colon 

bacillus,  92. 

Dextrose    broth,   compared    with 
lactose  bile,  148. 
broth,  as     preliminary    enrich- 
ment medium,  149. 
broth  tubes,  175. 
Diagnostic  characters  of  B.  coli, 

103. 

Dilution  for  colon  test,  95. 
Dilution  of  sample,  37. 
Dissociation,  effect  of,  19. 
Drigalski-Conradi  medium,  69. 
Driven  wells,  23. 
Dysentery  organisms  in  water,  85. 

Effect  of  dilution,  37. 
Eisner's  medium,  68. 
English  sewages,  188. 
Enrichment  methods,  67,  69. 
Enrichment    methods,    objections 

to,  68. 

Enumeration  of  bacteria,  25. 
Esmarch  tube,  44. 
Examination  of  large  samples,  94. 

of  springs,  1 80. 

of  water  supply  of  Hartford,  55. 

Filtering  action  of  soil,  22. 
Filtration,  efficiency  of,  52. 


Filtration,  mechanical,  53. 
"  Flaginac  coli,"  167. 
Food,  effect  of,  18. 

supply,  effect  of,  14,  20. 

supply,  effect  of,  20. 
Fuchsin  agar,  70. 

Gas  formation  with  B.  coli,  90. 

ratio,  atypical,  142. 

ratio  for  B.  coli,  105,  141. 
Gelatin,  phenolated,  68. 
Gelatin-plate  count,  175. 
Gelatin,  potato,  68. 
Gelatin  tube,  108. 
Glass,  effect  of  composition  of,  35. 
Glucose-formate  broth,  92. 
Green  plants,  N-requirements,  3. 
Ground  water  7. 
Ground-waters,  bacteria  in,  22. 
Ground-waters,  classes  of,  22. 

Hartford,   examination   of    water 

supply  of,  55. 
Hog  cholera  bacillus,  163. 
Hog  cholera  group,  164. 

Iceing,  effect  on  samples,  36. 
Impounding,  effect  of,  10. 
Increase  of  bacteria  in   Toronto 

water  supply,  55. 
Incubation,  41,  212. 
Incubation,  effect  of  period  of,  on 

count,  43. 
Incubator,  necessity  for  moisture 

in,  41. 
Incubator,   necessity    for    oxygen 

in,  41. 

Incubation,  period  of,  42. 
Incubation  temperature,  41. 
Indol  test,  108,  211. 


Subject  Index. 


Inorganic  constituents,  effect    of, 

19. 

Interpretation  of  results,  46. 
Intestinal  organisms,  59. 
Intestinal    bacteria   multiplication 

in  water,  21. 
Interpretation  of  results,  effect  of 

warm  weather  upon,  65. 
Isolation  of  B.  coli,  98. 
of  B.  sporogenes,  161. 
of  specific  pathogenes,  67. 


Lactic  acid  bacteria,  119. 

Lactose  bile,  147. 

Lactose  bile,   as  preliminary    en- 
richment medium,  149. 
bile,    compared    with    dextrose 

broth,  1 1 8. 
decomposition  of,  59. 
neutral  red  broth,  151. 

Lactose-bile,  use  of,  with  polluted 
waters,  92,  93. 

Large  samples,  examination  of,  94. 

Light,  effect  of,  14. 

Litmus-lactose-agar,  59. 

Litmus-lactose-agar    for    isolation 
of  B.  coli,  97. 

Litmus-lactose-agar  plate,  88. 

Litmus-lactose-agar    plates,   incu- 
bation of,  98. 

Litmus-lactose-agar,  possible  error 
with,  99. 

Litmus-lactose-agar,  use  of,  174. 

Malachite-green  agar,  70. 
Meat  infusion,  variability  of,  41. 
Mechanical  filters  compared  with 

sand  filters,  54. 
Mechanical  nitration,  53. 


Media,  preparation  of,  203,   208, 
209. 

ingredients  of,  203,  204. 

reaction  of,  205. 

sterilization  of,  204. 

storage  of,  206. 

Medium,    importance    of   compo- 
sition, 38,  39. 
Milk,  210. 

Minimal  nutrients,  49. 
Motility  of  B.  coli,  104. 

Nahrstoff  agar,  25,  26,  27. 
Nahrstoff -gelatin  ratio,  27. 
Nahrstoff  media,  effect  of,  26,  27. 
Neutral  red,  150. 

Neutral    red    broth,  as  presump- 
tive test,  150. 

Newport,  R.  I.,  typhoid  at,  55. 
Nitrate  solution,  210. 
Nitrates,  formation  by  bacteria,  4. 
Nitrification,  4. 
Nitrifying  organisms,  200. 
Nitrite  test,  108. 

Nitrites,  formation  by  bacteria,  4. 
Nitrogen,  cycle,  4,  5. 
Nitroso-indol  reaction,  83. 
"  Normal  inhabitants,"  58. 
Normal  waters,  49. 
Nutrients,  minimal,  49. 

Objectionable  aliens,  58. 
Ocean,  bacteria  in,  13. 
Organisms,  acid  forming,  60. 
Organisms,  intestinal,  59. 
Osmotic  pressure,  14. 
Overturns,  effect  of,  12. 
Ox  bile,  146. 

as  culture  medium,  146. 

as  presumptive  test,  147. 


256 


Subject  Index. 


Oxgyen,  effect  on  stored  samples, 

35- 

Oxygen,  effect  on  viability  of  bac- 
illi in  water,  18,  19. 

Paracolon  bacilli,   in,   116,   166, 

167. 

Paracolon  group,  127. 
Paratyphoid  bacilli,  163. 
Paratyphoid  organisms,  116. 
Parietti's  solution,  67. 
Pathogenes  in  water,  67. 
Peptone,  effect  of  kind  on  counts, 

39- 

Phenol  agar,  89.     t 
Phenolated  gelatin,  68. 
Plating,  procedure,  36,  37,  38. 
Polluted    waters,    acid    producers 

in,  60. 
Polluted  waters,  body  temperature 

count,  60. 

waters,  examination  of,  60. 
Porous  tops,  59. 
Potato  gelatin,  68. 
Precipitants  for  typhoid  isolation, 

75; 

Precipitation  methods,  69. 
Preliminary  cultivation,  213. 
Preliminary    enrichment    for     B. 

coli,  89. 
Presumptive  test,    correctness    of, 

144. 
test,   effect  of  temperature  on, 

145- 

test  in  absence  of  B.  coli,  145. 
test,  with  highly  polluted  waters, 

144. 

test,  interpretation,  143. 
tests,  176. 
tests  for  B.  coli,  141. 


Progressive  pollution,  179. 
Proteus  group,  168. 
Protozoa,  effect  of,  15. 
Pumping,  effect  of,  31. 

Quantitative  bacteriological  exam- 
ination, 25. 

Rain  water,  bacteria  in,  6. 

Ratio  of    acid    producers  to  total 

count,  64. 
Record  blank,  106. 
Recording  counts,  44. 
Red  colonies,  count  of,  175. 
Reduction    processes    in    sewage, 

201. 
Relation  of  total   count  to   body 

temperature  count,  61,  62,  63. 
Results,  interpretation  of,  46. 
River  waters,  bacteria  in,  8. 

Sample,  growth  of  bacteria  in,  33, 

34,  35- 
Sampling,  29. 

at  great  depths,  32. 
effect  of  size  of  bottle,  35. 
surface  waters,  32. 
Sand   niters    compared   with   me- 
chanical filters,  54. 
Sand  filtration,  52. 
Sanitary    analysis,    interpretation 

of,  183. 
Seasonal  distribution,  65. 

variation  of  bacteria  in  streams, 

7,  8. 

variation  in  reservoirs,  n. 
Sea-water,  bacteria  in,  13. 
Sedimentation,  14,  15. 
Self -purification,  19,  125. 
agencies,  14. 
in  Chicago  drainage  canal,  182. 


Subject  Index. 


257 


Sewage,   bacteriological   examina- 
tion of,  1 86. 

bacteriology  of,  184. 

determination  of  B.  coli  in,  187. 

disposal,  object  of,  184. 

diurnal    variation    in    bacterial 
content  of,  189. 

effluents,  bacteriology  of,  184. 

number  of  bacteria  in,  188. 

sterilization  of,  196,  197. 

streptococci,  152. 

streptococci,     agar     streak     of, 
100. 

streptococci,  overgrowth  by,  91. 
Shellfish,  185. 
Significance  of  B.  coli,  112. 
Snow,  bacteria  in,  6. 
Soil,  effect  of  depth  on  number  of 

bacteria,  22. 

Soil,  removal  of  bacteria  by,  22. 
Springs,  bacteria  in,  24. 
Spring  waters,  bacteria  in,  23. 
Standard  methods,  28. 

importance  of,  40, 

methods,  procedure,  97. 

methods,  test  for  B.  coli,  103. 

procedure,  66. 
Standards,  arbitrary  46,  47. 
Standing  water,  bacteria  in,  10. 
Staphylococci,  153. 
Storage,  effect  of,  10,  n. 
Streptococci,  99. 

agar  streak  of,  100. 

and  B.  coli,  detection  of,  158. 

and  B.  coli,  relation  to  sewage 
pollution,  158. 

and  B.  coli,  relative  growth  of, 
in  dextrose  broth,  156,  157. 

as  index  of  pollution,  153. 

fermenting  power,  160. 


Streptococci  —  Continued 

from  intestinal  tract,  160. 

in  polluted  rivers,  153, 

isolation  of,  155. 

significance  of,  175. 
Streptococcus  pyo genes,  152. 
Subsidence  of  bacteria,  14. 
Sugar  broths,  207. 
Sugar  agar,  209. 
Sunlight,  bactericidal  action  of,  17 

18. 

Sunlight,  effect  of,  17,  18. 
Surface  pollution,  effect  on  num- 
ber of  bacteria,  9,  10. 

wash,  7. 
Surface  water,  7. 

waters,  B.  coli  in,  130. 

Temperature,  effect  of,  14. 
effect  on  colon  bacilli,  17. 
effect     on      presumptive     test, 

145- 
relation  to  bacterial  life,  17. 

Thames  River,  B.  coli  in,  131. 

Thermophilic  bacteria,  185. 

Time,  effect  of,   on  self-purifica- 
tion, 21. 

Time,  effect  on  sample,  36. 

Toronto  water  supply,  increase  of 
bacteria  in,  55. 

Total  count,  57. 

Toxic  products,  effect  of,  16. 

Trickling  filters,  192. 

Tubular  wells,  23. 

Typhoid  bacilli,  direct  isolation  of, 

69. 
bacilli,     effect     of     dissociated 

hydrogen  on,  19. 
bacilli,  effect  of  other  organisms 
on  viability  of,  16. 


258 


Subject  Index. 


Typhoid  bacilli  —  Continued 
bacilli,  life  in  sewage,  16. 
bacilli,   life    in    sterile   sewage, 

16. 

bacilli,  life  in  water,  15,  16. 
bacilli,   number  of  in  polluted 

water,  82. 

bacilli,  persistence  in  mud,  21. 
bacilli,    persistence  in    sewage, 

21. 

bacilli,    persistence     in     water, 
21. 

growth  on  Drigalski-Conradi 

medium,  70. 
bacillus,  isolation  of,  69. 
bacillus,  isolation  with,  caffeine 

agar,   fuchsin  agar,  malchite- 

green  agar,  70. 
bacillus,  positive  isolation  from 

water,  80,  81. 

isolation  by  chemical  precipita- 
tion, 74,  75. 
at  Newport,  R.  I.,  55. 


Unobjectionable  aliens,  58. 
Unpolluted  waters,  body  temper- 
ature count  in,  60. 
Urea,  decomposition  of,  3. 

Value  of  Bacterial  analysis,  54. 
Variation    in    biochemical    tests, 
no. 

Warmth,  effect  on  colon  bacilli  in 

water,  17. 

Warm  weather,  effect  of,  on  inter- 
pretation of  results,  65. 
Waste  products,  effect  of,  37. 
Water    bacteria,    increase    during 

storage,  34,  35. 
Waters,  normal,  49. 
Wells,  B.  coli  in,  132,  133. 
pollution  of,  22,  23. 
bacteria  in,  23   52. 
deep,  23. 
Widal  test,  80. 


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8 


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9 


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10 


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11 


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12 


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Peele's  Compressed  Air  Plant  for  Mines 8vo,  3  00 

Poole's  Calorific  Power  of  Fuels 8vo,  3  00 

*  Porter's  Engineering  Reminiscences,  1855  to  1882 8vo,  3  00 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  00 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design. 8vo,  3  00 

Richards's  Compressed  Air 12mo,  1  50 

Robinson's  Principles  of  Mechanism. 8vo,  3  00 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  00 

Smith  (A.  W.)  and  Marx's  Machine  Design 8vo,  3  00 

Smith's  (O.)  Press-working  of  Metals 8vo,  3  00 

Sorel's  Carbureting  and  Combustion  in  Alcohol  Engines.      (Woodward  and 

Preston.) Large  12mo,  3  00 

Stone's  Practical  Testing  of  Gas  and  Gas  Meters 8vo,  3  50 

13 


Thurston's  Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics. 

12mo,  $1  00 

Treatise  on  Friction  and  Lost  Work  in  Machinery  and  Mill  Work.  .  .8vo,  3  00 

*  Tillson's  Complete  Automobile  Instructor 16mo,  1  50 

*  Titsworth's  Elements  of  Mechanical  Drawing Oblong  8vo,  1  25 

Warren's  Elements  of  Machine  Construction  and  Drawing 8vo,  7  50 

*  Waterbury's  Vest  Pocket  Hand-book  of  Mathematics  for  Engineers. 

2JX5I  inches,  mor.  1   00 
Weisbach's    Kinematics    and    the    Power   of   Transmission.     (Herrmann — 

Klein.) 8vo,  5  00 

Machinery  of  Transmission  and  Governors.      (Hermann — Klein.).  .8vo,  5  00 

Wood's  Turbines 8vo,  2  50 


MATERIALS   OF   ENGINEERING. 

*  Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering 8vo,  7  50 

Church's  Mechanics  of  Engineering 8vo,  6  00 

*  Greene's  Structural  Mechanics 8vo,  2  50 

*  Holley's  Lead  and  Zinc  Pigments Large  12mo  3  00 

Holley  and  Ladd's  Analysis  of  Mixed  Paints,  Color  Pigments,  and  Varnishes. 

Large  12mo,  2  50 
Johnson's  (C.  M.)  Rapid    Methods    for    the    Chemical    Analysis    of    Special 

Steels,  Steel-Making  Alloys  and  Graphite Large  12mo,  3  00 

Johnson's  (J.  B.)  Materials  of  Construction 8vo,  6  00 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Maire's  Modern  Pigments  and  their  Vehicles 12mo,  2  00 

Martens's  Handbook  on  Testing  Materials.      (Henning.) 8vo,  7  50 

Maurer's  Techincal  Mechanics 8vo,  4  00 

Merriman  s  Mechanics  of  Materials 8vo,  5  00 

*  Strength  of  Materials 12mo,  1  00 

Metcalf's  Steel.     A  Manual  for  Steel-users 12mo,  2  00 

Sabin's  Industrial  and  Artistic  Technology  of  Paint  and  Varnish 8vo,  3  00 

Smith's  ((A.  W.)  Materials  of  Machines 12mo,  1  00 

Smith's  (H.  E.)  Strength  of  Material 12mo, 

Thurston's  Materials  of  Engineering 3  vols.,  8vo,  8  00 

Part  I.      Non-metallic  Materials  of  Engineering, 8vo,  2  00 

Part  II.     Iron  and  Steel 8vo,  3  50 

Part  III.      A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Wood's  (De  V.)  Elements  of  Analytical  Mechanics 8vo,  3  00 

Treatise  on    the    Resistance    of    Materials    and    an    Appendix    on    the 

Preservation  of  Timber 8vo,  2  00 

Wood's  (M.  P.)  Rustless  Coatings:    Corrosion  and  Electrolysis  of  Iron  and 

Steel 8vo,  4  00 


STEAM-ENGINES    AND    BOILERS. 

Berry's  Temperature-entropy  Diagram 12mo,  2  00 

Carnot's  Reflections  on  the  Motive  Power  of  Heat.      (Thurston.) 12mo,  1   50 

Chase's  Art  of  Pattern  Making 12mo,  2  50 

Creighton's  Steam-engine  and  ether  Heat  Motors 8vo,  5  00 

Dawson's  "'Engineering''  and  Electric  Traction  Pocket-book.  ..  .  16mo,  mor.  5  00 

Ford's  Boiler  Making  for  Boiler  Makers 18mo,  1  00 

*  Gebhardt's  Steam  Power  Plant  Engineering 8vo,  6  00 

Goss's  Locomotive  Performance 8vo,  5  00 

Hemenway's  Indicator  Practice  and  Steam-engine  Economy 12mo,  2  00 

Button's  Heat  and  Heat-engines 8vo.  5  00 

Mechanical  Engineering  of  Power  Plants 8vo,  5  00 

Kent's  Steam  boiler  Economy 8vo,  4  00 

14 


Kneass's  Practice  and  Theory  of  the  Injector 8vo,  $1  50 

MacCord's  Slide-valves 8vo,  2  00 

Meyer's  Modern  Locomotive  Construction 4to,  10  00 

Moyer's  Steam  Turbine 8vo,  4  00 

Peabody's  Manual  of  the  Steam-engine  Indicator 12mo,  1  50 

Tables  of  the  Properties  of  Steam  and  Other  Vapors  and  Temperature- 
Entropy  Table 8vo,  1  00 

Thermodynamics  of  the  Steam-engine  and  Other  Heat-engines.  .  .  .8vo,  5  00 

Valve-gears  for  Steam-engines 8vo,  2  50 

Peabody  and  Miller's  Steam-boilers 8vo,  4  00 

Pupin's  Thermodynamics  of  Reversible  Cycles  in  Gases  and  Saturated  Vapors. 

(Osterberg.) 12mo,  1   25 

Reagan's  Locomotives:  Simple,  Compound,  and  Electric.     New  Edition. 

Large  12mo,  3  50 

Sinclair's  Locomotive  Engine  Running  and  Management 12mo,  2  00 

Smart's  Handbook  of  Engineering  Laboratory  Practice 12mo,  2  50 

Snow's  Steam-boiler  Practice 8vo,  3  00 

Spangler's  Notes  on  Thermodynamics 12mo,  1  00 

Valve-gears 8vo,  2  50 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo,  3  00 

Thomas's  Steam-turbines 8vo,  4  00 

Thurston's  Handbook  of  Engine  and  Boiler  Trials,  and  the  Use  of  the  Indi- 
cator and  the  Prony  Brake 8vo,  5  00 

Handy  Tables 8vo,  1  50 

Manual  of  Steam-boilers,  their  Designs,  Construction,  and  Operation  8vo,  5  00 

Manual  of  the  Steam-engine 2vols.,   8vo,  10  00 

Part  I.     History,  Structure,  and  Theory 8vo,  6  00 

Part  II.      Design,  Construction,  and  Operation 8vo,  6  00 

Steam-boiler  Explosions  in  Theory  and  in  Practice 12mo,  1  50 

Wehrenfennig's    Analysis  and  Softening  of  Boiler  Feed- water.     (Patterson). 

8vo,  4  00 

Weisbach's  Heat,  Steam,  and  Steam-engines.      (Du  Boie.) 8vo,  5  00 

Whitham's  Steam-engine  Design 8vo,  5  00 

Wood's  Thermodynamics,  Heat  Motors,  and  Refrigerating  Machines.  .  ,8vo,  4  00 


MECHANICS    PURE   AND    APPLIED. 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Notes  and  Examples  in  Mechanics 8vo,  2  00 

Dana's  Text-book  of  Elementary  Mechanics  for  Colleges  and  Schools  .12mo,  1  50 
Du  Bois's  Elementary  Principles  of  Mechanics: 

Vol.    I.     Kinematics 8vo,  3  50 

Vol.  II.     Statics 8vo,  4  00 

Mechanics  of  Engineering.     Vol.     I Small  4to,  7  50 

Vol.  II Small  4to,  10  00 

*  Greene's  Structural  Mechanics 8vo,  2  50 

James's  Kinematics  of  a  Point  and  the  Rational  Mechanics  of  a  Particle. 

Large  12mo,  2  00 

*  Johnson's  (W.  W.)  Theoretical  Mechanics.  : 12mo,  3  00 

Lanza's  Applied  Mechanics 8vo,  7  50 

*  Martin's  Text  Book  on  Mechanics,  Vol.  I,  Statics 12mo,  1  25 

*  Vol.  II,  Kinematics  and  Kinetics.  12mo,  1  50 

Maurer's  Technical  Mechanics 8vo,  4  00 

*  Merriman's  Elements  of  Mechanics 12mo,  1  00 

Mechanics  of  Materials 8vo,  5  00 

*  Michie's  Elements  of  Analytical  Mechanics 8vo,  4  00 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

Sanborn's  Mechanics  Problems Large  12mo,  1  50 

Schwamb  and  Merrill's  Elements  of  Mechanism 8vo,  3  00 

Wood's  Elements  of  Analytical  Mechanics 8vo,  3  00 

Principles  of  Elementary  Mechanics 12mo,  1  25 


15 


MEDICAL. 

*  Abderhalden's  Physiological   Chemistry  in   Thirty   Lectures.     (Hall   and 

Defren.) 8vo,  $5  00 

von  Behring's  Suppression  of  Tuberculosis.      (Bolduan.) 12mo,  1  BO 

Bolduan's  Immune  Sera 12mo,  1  50 

Bordet's  Studies  in  Immunity.     (Gay).     (In  Press.) .8vo, 

Davenport's  Statistical  Methods  with  Special  Reference  to  Biological  Varia- 
tions  16mo,  mor.  1  50 

Ehrlich's  Collected  Studies  on  Immunity.     (Bolduan.) 8vo,  6  00 

*  Fischer's  Physiology  of  Alimentation Large  12mo,  2  09 

de  Fursac's  Manual  of  Psychiatry.      (Rosanoff  and  Collins.)..  .  .Large  12mo,  2  50 

Hammarsten's  Text-book  on  Physiological  Chemistry.      (Mandel.) 8vo,  4  00 

Jackson's  Directions  for  Laboratory  Work  in  Physiological  Chemistry.  .8vo,  1  25 

Lassar-Cohn's  Practical  Urinary  Analysis.      (Lorenz.) 12mo,  1  00 

Mandel's  Hand-book  for  the  Bio-Chemical  Laboratory 12mo,  1  50 

*  Pauli's  Physical  Chemistry  in  the  Service  of  Medicine.      (Fischer.)  ..12mo,  1  25 

*  Pozzi-Escot's  Toxins  and  Venoms  and  their  Antibodies.      (Cohn.).  .  12mo,  1  00 

Rostoski's  Serum  Diagnosis.      (Bolduan.) 12mo,  1  00 

Ruddiman's  Incompatibilities  in  Prescriptions 8vo,  2  00 

Whys  in  Pharmacy 12mo,  1  00 

Salkowski's  Physiological  and  Pathological  Chemistry.     (Orndorff.)  ....8vo,  2  50 

*  Satterlee's  Outlines  of  Human  Embryology 12mo,  1  25 

Smith's  Lecture  Notes  on  Chemistry  for  Dental  Students 8vo,  2  50 

*  Whipple's  Tyhpoid  Fever Large  12mo,  3  00 

Woodhull's  Notes  on  Military  Hygiene 16mo,  1  50 

*  Personal  Hygiene 12mo,  1  00 

Worcester  and  Atkinson's  Small  Hospitals  Establishment  and  Maintenance, 
and  Suggestions  for  Hospital  Architecture,  with  Plans  for  a  Small 

Hospital 12mo,  1  25 


METALLURGY. 

Betts's  Lead  Refining  by  Electrolysis 8vo,  4  09 

Bolland's  Encyclopedia  of  Founding  and  Dictionary  of  Foundry  Terms  used 

in  the  Practice  of  Moulding 12mo,  3  00 

Iron  Founder 12mo,  2  50 

Supplement 12mo,  2  50 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12mo,  1  00 

Goesel's  Minerals  and  Metals:  A  Reference  Book 16mo,  mor.  3  00 

*  Iles's  Lead-smelting 12mo,  2  50 

Johnson's    Rapid    Methods   for    the   Chemical   Analysis   of   Special   Steels, 

Steel-making  Alloys  and  Graphite Large  12mo,  3  00 

Keep's  Cast  Iron 8vo,  2  50 

Le  Chatelier's  High- temperature  Measurements.     (Boudouard — Burgess.) 

12mo,  3  00 

Metcalf's  Steel.      A  Manual  for  Steel-users 12mo,  2  00 

Minet's  Production  of  Aluminum  and  its  Industrial  Use.      (Waldo.).  .  12mo,  2  50 

Ruer's  Elements  of  Metallography.      (Mathewson) 8vo, 

Smith's  Materials  of  Machines « 12mo,  1  00 

Tate  and  Stone's  Foundry  Practice 12mo,  2  00 

Thurston's  Materials  of  Engineering.      In  Three  Parts 8vo,  8  00 

Part  I.       Non-metallic  Materials  of  Engineering,  see  Civil  Engineering, 
page  9. 

Part  II.     Iron  and  Steel 8vo,  3  50 

Part  III.  A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo,  2  50 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo,  3  00 

West's  American  Foundry  Practice 12mo,  2  50 

Moulders'  Text  Book 12mo,  2  50 

16 


MINERALOGY. 

Baskerville's  Chemical  Elements.     (In  Preparation.). 

Boyd's  Map  of  Southwest  Virginia Pocket-book  form.  $2  00 

*  Browning's  Introduction  to  the  Rarer  Elements 8vo,  1  50 

Brush's  Manual  of  Determinative  Mineralogy.      (Penfield.) 8vo,  4  00 

Butler's  Pocket  Hand-book  of  Minerals 16mo,  mor.  3  00 

Chester's  Catalogue  of  Minerals 8vo,  paper,     1  00 

Cloth,  1  25 

*  Crane's  Gold  and  Silver 8vo,  5  00 

Dana's  First  Appendix  to  Dana's  New  "System  of  Mineralogy".  .Large  8vo,  1  00 
Dana's  Second  Appendix  to  Dana's  New  "  System  of  Mineralogy." 

Large  8vo, 

Manual  of  Mineralogy  and  Petrography 12mo,  2  00 

Minerals  and  How  to  Study  Them 12mo,  1  50 

System  of  Mineralogy Large  8vo,  half  leather,  12  50 

Text-book  of  Mineralogy 8vo,  4  00 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12mo,  1  00 

Eakle's  Mineral  Tables 8vo,  1  25 

Eckel's  Stone  and  Clay  Products  Used  in  Engineering.      (In  Preparation). 

Goesel's  Minerals  and  Metals:  A  Reference  Book 16mo,  mor.  3  00 

Groth's  Introduction  to  Chemical  Crystallography  (Marshall) 12mo,  1  25 

*  Hayes's  Handbook  for  Field  Geologists 16mo,  mor.  1  50 

Iddings's  Igneous  Rocks 8vo,  5  00 

Rock  Minerals 8vo,  5  00 

Johannsen's  Determination  of  Rock-forming  Minerals  in  Thin  Sections.  8vo, 

With  Thumb  Index  5  00 

*  Martin's  Laboratory    Guide    to    Qualitative    Analysis    with    the    Blow- 

pipe  12mo,  60 

Merrill's  Non-metallic  Minerals.  Their  Occurrence  and  Uses 8vo,  4  00 

Stones  for  Building  and  Decoration 8vo.  5  00 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  50 
Tables  of  Minerals,    Including  the  Use  of  Minerals  and  Statistics  of 

Domestic  Production 8vo.  1  00 

*  Pirsson's  Rocks  and  Rock  Minerals 12mo,  2  50 

*  Richards's  Synopsis  of  Mineral  Characters 12mo,  mor.  1  25 

*  Ries's  Clays:  Their  Occurrence,  Properties  and  Uses 8vo.  5  00 

*  Ries  and  Leighton's  History  of  the  Clay-working  Industry  of  the  United 

States 8vo,  2  50 

*  Tillman's  Text-book  of  Important  Minerals  and  Rocks 8vo.  2  09 

Washington's  Manual  of  the  Chemical  Analysis  of  Rocks 8vo,  2  00 


MINING. 

*  Beard's  Mine  Gases  and  Explosions , Large  12mo,  3  00 

Boyd's  Map  of  Southwest  Virginia Pocket-book  form,  2  00 

*  Crane's  Gold  and  Silver 8vo  5  00 

*  Index  of  Mining  Engineering  Literature 8vo.  4  00 

*  8vo.  mor.  5  00 

Douglas's  Untechnical  Addresses  on  Technical  Subjects 12mo.  1  00 

Eissler's  Modern  High  Explosives 8vo.  4  00 

Goesel's  Minerals  and  Metals:  A  Reference  Book 16mo.  mor.  3  00 

Ihlseng's  Manual  of  Mining 8vo,  5  00 

*  Iles's  Lead  Smelting .  I2mo.  2  50 

Peele's  Compressed  Air  Plant  for  Mines 8vo.  3  00 

Riemer's  Shaft  Sinking  Under  Difficult  Conditions.     (Corning  and  Peele).8vo,  3  00 

*  Weaver's  Military  Explosives gvo,  3  00 

Wilson's  Hydraulic  and  Placer  Mining.     2d  edition   rewritten 12mo,  2  50 

Treatise  on  Practical  and  Theoretical  Mine  Ventilation 12mo.  1  25 

17 


SANITARY    SCIENCE. 

Association  of  State  and  National  Food  and  Dairy  Departments,  Hartford 

Meeting,  1906 8vo,  $3  00 

Jamestown  Meeting,  1907 8vo,  3  00 

*  Bashore's  Outlines  of  Practical  Sanitation 12mo,  1  23 

Sanitation  of  a  Country  House 12mo,  1  00 

Sanitation  of  Recreation  Camps  and  Parks 12mo,  1  00 

Folwell's  Sewerage.      (Designing,  Construction,  and  Maintenance.) 8vo,  3  00 

Water-supply  Engineering 8vo,  4  00 

Fowler's  Sewage  Works  Analyses 12mo,  2  00 

Fuertes's  Water-filtration  Works 12mo,  2  50 

Water  and  Public  Health 12mo,  1  50 

Gerhard's  Guide  to  Sanitary  Inspections 12mo,  1  50 

*  Modern  Baths  and  Bath  Houses 8vo,  3  00 

Sanitation  of  Public  Buildings 1 2mo,  1  50 

Hazen's  Clean  Water  and  How  to  Get  It Large  12mo,  1  50 

Filtration  of  Public  Water-supplies 8vo,  3  00 

Kinnicut,  Winslow  and  Pratt's  Purification  of  Sewage.      (In  Preparation.) 
Leach's  Inspection  and  Analysis  of  Food  with  Special  Referencj  to  State 

Control „ 8vo,  7  50 

Mason's  Examination  of  Water.     (Chemical  and  Bacteriological) 12mo,  1  25 

Water-supply.      (Considered  principally  from  a  Sanitary  Standpoint). 

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*  Merriman's  Elements  of  Sanitary  Enigneering 8vo,  2  00 

Ogden's  Sewer  Construction 8vo,  3  00 

Sewer  Design 12mo,  2  00 

Parsons's  Disposal  of  Municipal  Refuse 8vo,  2  00 

Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Refer- 
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*  Price's  Handbook  on  Sanitation 12mo,  1   50 

Richards's  Cost  of  Cleanness 12mo,  1  00 

Cost  of  Food.     A  Study  in  Dietaries 12mo,  1  00 

Cost  of  Living  as  Modified  by  Sanitary  Science 12mo,  1  00 

Cost  of  Shelter 12mo,  1  00 

*  Richards  and  Williams's  Dietary  Computer 8vo,  1  50 

Richards  and  Woodman's  Air,   Water,   and  Food  from  a  Sanitary  Stand- 
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*  Richey's     Plumbers',     Steam-fitters',    and     Tinners'     Edition     (Building 

Mechanics'  Ready  Reference  Series) 16mo,  mor.  1  50 

Rideal's  Disinfection  and  the  Preservation  of  Food 8vo,  4  00 

Sewage  and  Bacterial  Purification  of  Sewage 8vo,  4  00 

Soper's  Air  and  Ventilation  of  Subways 12mo,  2  50 

Turneaure  and  Russell's  Public  Water-supplies 8vo,  5  00 

Venable's  Garbage  Crematories  in  America 8vo,  2  00 

Method  and  Devices  for  Bacterial  Treatment  of  Sewage 8vo,  3  00 

Ward  and  Whipple's  Freshwater  Biology.      (In  Press.) 

Whipple's  Microscopy  of  Drinking-water 8vo,  3  50 

*  Typhoid  Fever Large  12mo,  3  00 

Value  of  Pure  Water Large  12mo,  1  00 

Winslow's  Systematic  Relationship  of  the  Coccacese Large  12mo,  2  50 


MISCELLANEOUS. 

Emmons's  Geological  Guide-book  of  the  Rocky  Mountain  Excursion  of  the 

International  Congress  of  Geologists .Large  8vo.  1   50 

Ferrel's  Popular  Treatise  on  the  Winds 8vo,  4  00 

Fitzgerald's  Boston  Machinist 18mo,  1  00 

Gannett's  Statistical  Abstract  of  the  World 24mo,  75 

Haines's  American  Railway  Management.  .  .  . 12mo,  2  50 

Hanausek's  The  Microscopy  of  Technical  Products.     (Winton) 8vo,  5  00 

18 


Jacobs's  Betterment    Briefs.     A    Collection    of    Published    Papers    on    Or- 
ganized Industrial  Efficiency 8vo,  $3  50 

Metcalfe's  Cost  of  Manufactures,  and  the  Administration  of  Workshops.. 8vo,  5  00 

Putnam's  Nautical  Charts. 8vo,  2  OO 

Ricketts's  History  of  Rensselaer  Polytechnic  Institute  1824-1894. 

Large  12mo,  3  OO 

Rotherham's  Emphasised  New  Testament Large  8vo,  2  00 

Rust's  Ex-Meridian  Altitude,  Azimuth  and  Star-finding  Tables 8vo,  5  00 

Standage's  Decoration  of  Wood,  Glass,  Metal,  etc.  .  . 12mo,  2  00 

Thome's  Structural  and  Physiological  Botany.     (Bennett) 16mo,  2  25 

Westermaier's  Compendium  of  General  Botany.     (Schneider) 8vo,  2  00 

Winslow's  Elements  of  Applied  Microscopy 12mo,  1 .50 


HEBREW   AND   CHALDEE   TEXT-BOOOKS. 

Gesenius's  Hebrew  and  Chaldee  Lexicon  to  the  Old  Testament  Scriptures. 

(Tregelles.) Small  4to,  half  mor,     5  00 

Green's  Elementary  Hebrew  Grammar 12mo,      1  25 


19 


Engineering 


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Engineering 


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